Compositions and methods of use for treating metabolic disorders

ABSTRACT

Methods of treating individuals with a glucose metabolism disorder and/or a body weight disorder, and compositions associated therewith, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority benefit of U.S. provisional applicationSer. No. 61/758,456, filed Jan. 30, 2013, and of U.S. provisionalapplication Ser. No. 61/882,542, filed Sep. 25, 2013, each of whichapplications is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to, among other things, growthdifferentiation factor muteins and modifications thereof which areuseful in treating obesity, diabetes and other metabolic-relateddisorders.

BACKGROUND

Obesity is most commonly caused by excessive food intake coupled withlimited energy expenditure and/or lack of physical exercise. Obesityincreases the likelihood of development of various diseases, such asdiabetes mellitus, hypertension, atherosclerosis, coronary arterydisease, sleep apnea, gout, rheumatism and arthritis. Moreover,mortality risk directly correlates with obesity, such that, for example,a body-mass index in excess of 40 results in an average decreased lifeexpectancy of more than 10 years.

Current pharmacological treatment modalities include appetitesuppressors targeting receptor classes (e.g., CB1, 5-HT_(2C), and NPY);regulators of the appetite circuits in the hypothalamus and themolecular actions of ghrelin; and nutrient-absorption inhibitorstargeting lipases. Unfortunately, none of the current modalities hasbeen shown to effectively treat obesity without causing adverse effects,some of which can be very severe.

High blood glucose levels stimulate the secretion of insulin bypancreatic beta-cells. Insulin in turn stimulates the entry of glucoseinto muscles and adipose cells, leading to the storage of glycogen andtriglycerides and to the synthesis of proteins. Activation of insulinreceptors on various cell types diminishes circulating glucose levels byincreasing glucose uptake and utilization, and by reducing hepaticglucose output. Disruptions within this regulatory network can result indiabetes and associated pathologic syndromes that affect a large andgrowing percentage of the human population.

Patients who have a glucose metabolism disorder can suffer fromhyperglycemia, hyperinsulinemia, and/or glucose intolerance. An exampleof a disorder that is often associated with the aberrant levels ofglucose and/or insulin is insulin resistance, in which liver, fat, andmuscle cells lose their ability to respond to normal blood insulinlevels.

In view of the prevalence and severity of obesity, diabetes andassociated metabolic and non-metabolic disorders, along with theshortcomings of current treatment options, alternative treatmentmodalities that modulate, for example, appetite, glucose and/or insulinlevels and enhance the biological response to fluctuating glucose levelsin a patient remain of interest.

In addition, in the pharmaceutical sciences it is frequently beneficial,and sometimes imperative, to improve one of more physical properties ofthe treatment modality (e.g., a protein, peptide, or hydrophobicmolecule) of interest and/or the manner in which it is administered.Improvements of physical properties include, for example, methods ofincreasing water solubility, bioavailability, serum half-life, and/ortherapeutic half-life; modulating immunogenicity and/or biologicalactivity; and/or extending the circulation time. Such improvements mustbe imparted without adversely impacting the bioactivity of the treatmentmodality. Thus, it may be advantageous for alternatives to currenttreatment options for obesity, diabetes and associated metabolic andnon-metabolic disorders, to possess one or more improved physicalproperties.

SUMMARY

The present disclosure contemplates the use of the agents describedherein, and compositions thereof, to treat and/or prevent variousdiseases, disorders and conditions, and/or the symptoms thereof. In someembodiments, the diseases, disorders and conditions, and/or the symptomsthereof, relate to glucose metabolism disorders and othermetabolic-related disorders, whereas in other embodiments they relate tobody weight disorders. By way of example, but not limitation, theagents, and compositions thereof, can be used for the treatment and/orprevention of diabetes mellitus (e.g., Type 2 diabetes), insulinresistance and diseases, disorders and conditions characterized byinsulin resistance, decreased insulin production, hyperglycemia,hypoinsulinemia, and metabolic syndrome. The agents, and compositionsthereof, can also be used for the treatment and/or prevention of obesityand other body weight disorders by, for example, effecting appetitesuppression.

In certain embodiments, the agents are human Growth DifferentiationFactor 15 (GDF15)-related polypeptides, and homologues, variants (e.g.,muteins), fragments and other modified forms thereof. In particularembodiments, the agents contemplated by the present disclosure aremodified human GDF15 molecules, whereas in other embodiments the agentsare modified GDF15 muteins. The present disclosure also contemplatesnucleic acid molecules encoding the foregoing. For the sake ofconvenience, the modified human GDF15 molecules and the modified GDF15variants (e.g., muteins) described henceforward are collectivelyreferred to hereafter as the “Polypeptide(s)”. It should be noted thatany reference to “human” in connection with the polypeptides and nucleicacid molecules of the present disclosure is not meant to be limitingwith respect to the manner in which the polypeptide or nucleic acid isobtained or the source, but rather is only with reference to thesequence as it may correspond to a sequence of a naturally occurringhuman polypeptide or nucleic acid molecule. In addition to the humanpolypeptides and the nucleic acid molecules which encode them, thepresent disclosure contemplates GDF15-related polypeptides andcorresponding nucleic acid molecules from other species.

The present disclosure also contemplates other GDF15-related agentscapable of eliciting a biological response comparable to (or greaterthan) that of the Polypeptides, and/or agents capable of enhancing theactivity of the Polypeptides.

In some embodiments of the present disclosure, a subject having, or atrisk of having, a disease or disorder treatable by one or morePolypeptides is administered in an amount effective for treating thedisease or disorder. In some embodiments, the disease or disorder is ahyperglycemic condition, insulin resistance, hyperinsulinemia, glucoseintolerance or metabolic syndrome. In other embodiments the disease ordisorder is a body weight disorder (e.g., obesity), while in still otherembodiments the Polypeptides cause, to at least some extent, appetitesuppression.

Other aspects of the present disclosure include cell-based expressionsystems, vectors, engineered cell lines, and methods and uses related tothe foregoing.

As described in detail hereafter, one embodiment of the presentdisclosure relates to a polypeptide comprising a) a polypeptidecomprising at least one modification to the sequence depicted in FIG. 1B(SEQ ID NO:3); wherein the modification does not alter the amino acidsequence of the polypeptide, or b) a mutein polypeptide of the sequencedepicted in FIG. 1B (SEQ ID NO:3), wherein the mutein polypeptidecomprises at least one modification that does not alter the amino acidsequence of the mutein polypeptide; and wherein the modification setforth in a) and b) improves at least one physical property of thepolypeptide or the mutein polypeptide.

In certain embodiments of the present disclosure, a polypeptidecomprises a mutein polypeptide of any one of the sequences depicted in,for example, FIGS. 3, 5, 6 and 13.

In some embodiments, the polypeptide has a length of from about 10 aminoacids to about 113 amino acids. In other embodiments, a polypeptide ofthe present disclosure may have fewer than 100 amino acid residues,fewer than 75 amino acid residues, fewer than 50 amino acid residues,fewer than 25 amino acid residues, or fewer than 20 amino acid residues.

In still further embodiments, a polypeptide of the present disclosurecomprises an amino acid sequence having at least 85% amino acididentity, at least 90% amino acid identity, at least 93% amino acididentity, at least 95% amino acid identity, at least 97% amino acididentity, at least 98% amino acid identity, or at least 99% amino acididentity to the amino acid sequence depicted in FIG. 1B (SEQ ID NO:3)

According to the present disclosure, the polypeptide may be producedrecombinantly.

In some embodiments of the present disclosure, the modification to aPolypeptide comprises pegylation, glycosylation, polysialylation,hesylation, albumin fusion, albumin binding through a conjugated fattyacid chain, Fc-fusion, or fusion with a PEG mimetic.

In particular embodiments, the modification to a polypeptide comprisesglycosylation, and in some of those embodiments the glycosylation isN-glycosylation. The N-glycosylation may occur at more than one aminoacid residue of the polypeptide.

In other embodiments, the modification to a polypeptide comprises analbumin fusion wherein an albumin, an albumin variant, or an albuminfragment is conjugated to the polypeptide. In some embodiments, thealbumin, albumin variant, or albumin fragment is human serum albumin(HSA), a human serum albumin variant, or a human serum albumin fragment,whereas in other embodiments the albumin, albumin variant, or albuminfragment is bovine serum albumin, a bovine serum albumin variant, or abovine serum albumin fragment.

The full-length HSA has a signal peptide of 18 amino acids(MKWVTFISLLFLFSSAYS; SEQ ID NO:164) followed by a pro-domain of 6 aminoacids (RGVFRR; SEQ ID NO: 165); this 24 amino acid residue peptide maybe referred to as the pre-pro domain. The mature HSA polypeptide spansresidues D25-L609 of the sequence depicted in FIG. 1C (SEQ ID NO:5). Ina construct used to generate the experimental data presented herein, theendogenous signal peptide was replaced with human IgK signal peptide,and the endogenous pro-domain was left out entirely.

In still further embodiments, the albumin, albumin variant, or albuminfragment is conjugated to the polypeptide at the carboxyl terminus, theamino terminus, both the carboxyl and amino termini, or internally.Particular embodiments entail conjugation of the albumin, albuminvariant, or albumin fragment to the polypeptide at the amino terminus.

In particular embodiments, the albumin, albumin variant, or albuminfragment is conjugated to a polypeptide comprising the 167 amino acidpro-domain and the 112 amino acid mature domain of the 308 amino acidGDF15 precursor polypeptide; thus, the present disclosure contemplates aGDF15 polypeptide that has a length of from about amino acid residue 30to about amino acid residue 308 of the sequence depicted in FIG. 1A (SEQID NO:1).

The present disclosure contemplates direct expression and production ofthe 112 amino acid mature domain of GDF15 as depicted in FIG. 1B (SEQ IDNO:3), absent the 167 amino acid pro-domain, using a signal peptide ofappropriate length to confer secretion from mammalian tissue culture. Anexample of a suitable signal peptide to facilitate expression andsecretion includes IgK. The art describes mechanisms by which otherappropriate signal peptides can be identified [see, e.g., Ng et al.(January 2013) “Engineering Signal Peptides for Enhanced ProteinSecretion from Lactococcus lactis.”, Appl. Environ. Microbiol.79(1):347-56; Chou (2001) “Using Subsite Coupling to Predict SignalPeptides”, Protein Engineering 14(2):75-79; Leversen et al. (July 2009)“Evaluation of Signal Peptide Prediction Algorithms for Identificationof Mycobacterial Signal Peptides Using Sequence Data from ProteomicMethods” Microbiology 155(7):2357-83; and Shen et al., (2007)“Signal-3L: a 3-layer Approach for Predicting Signal Peptides”,Biochemical and Biophysical Res. Comm. 363: 297-303].

The present disclosure contemplates albumin fusion molecules wherein thealbumin, albumin variant, or albumin fragment is conjugated to thepolypeptide via a linker. Examples of suitable linkers are describedherein. By way of example, the linker may be a peptide linker of, forexample, four-to-six amino acids. In some embodiments, the linker is anon-cleavable linker (e.g., a 3×(4Gly-Ser) linker; SEQ ID NO:64). Inother embodiments, the linker is a cleavable linker, and in furtherembodiments the cleavable linker can be cleaved by a protease (e.g., a2×(4Gly-Ser) Factor Xa-cleavable linker (GGGGSGGGGSIXGR where X can beeither E or D (SEQ ID NO:221)).

In particular embodiments, the albumin, albumin variant, or albuminfragment of an albumin fusion molecule is excised prior to the albuminfusion molecule being secreted from a cell, whereas in other embodimentsthe albumin fusion molecule is excised subsequent to the albumin fusionmolecule being secreted from a cell.

The present disclosure encompasses embodiments wherein the physicalproperty of the recited polypeptide is selected from the groupconsisting of solubility, bioavailability, serum half-life, therapeutichalf-life, circulation time, and immunogenicity. In particularembodiments, the physical property is solubility.

Furthermore, the present disclosure contemplates nucleic acid moleculesencoding the aforementioned polypeptides. In some embodiments, a nucleicacid molecule is operably linked to an expression control element thatconfers expression of the nucleic acid molecule encoding the polypeptidein vitro, in a cell or in vivo.

In some embodiments, a vector (e.g., a viral vector) contains one ormore of the nucleic acid molecules.

Some embodiments include transformed or host cells that express one ormore of the aforementioned polypeptides.

In particular embodiments of the present disclosure, one or more of theaforementioned polypeptides is formulated to yield a pharmaceuticalcomposition, wherein the composition also includes one or morepharmaceutically acceptable diluents, carriers or excipients. In certainembodiments, a pharmaceutical composition also includes at least oneadditional prophylactic or therapeutic agent.

Still further embodiments of the present disclosure comprise an antibodythat binds specifically to one of the aforementioned muteinpolypeptides. In some embodiments, the antibody comprises a light chainvariable region and a heavy chain variable region present in separatepolypeptides or in a single polypeptide. An antibody of the presentdisclosure binds the polypeptide with an affinity of from about 10⁷ M⁻¹to about 10¹² M⁻¹ in certain embodiments. In still other embodiments,the antibody comprises a heavy chain constant region of the isotypeIgG1, IgG2, IgG3, or IgG4. In additional embodiments, the antibody isdetectably labeled, while it is a Fv, scFv, Fab, F(ab′)₂, or Fab′ inother embodiments.

The present disclosure also contemplates antibodies that comprise acovalently linked non-polypeptide polymer (e.g., a poly(ethylene glycol)polymer). In other embodiments, the antibody comprises a covalentlylinked moiety selected from a lipid moiety, a fatty acid moiety, apolysaccharide moiety, and a carbohydrate moiety.

The antibody is a single chain Fv (scFv) antibody in some embodiments,and the scFv is multimerized in others.

The antibodies of the present disclosure may be, but are not limited to,monoclonal antibodies, polyclonal antibodies, or humanized antibodies.

Furthermore, the present disclosure contemplates pharmaceuticalcompositions comprising an antibody as described above formulated withat least one pharmaceutically acceptable excipient, carrier or diluent.Such pharmaceutical compositions may also contain at least oneadditional prophylactic or therapeutic agent.

Certain embodiments of the present disclosure contemplate a sterilecontainer that contains one of the above-mentioned pharmaceuticalcompositions and optionally one or more additional components. By way ofexample, but not limitation, the sterile container may be a syringe. Instill further embodiments, the sterile container is one component of akit; the kit may also contain, for example, a second sterile containerthat contains at least one prophylactic or therapeutic agent.

The present disclosure also contemplates a method of treating orpreventing a glucose metabolism disorder in a subject (e.g., a human) byadministering to the subject a therapeutically effective amount of apolypeptide. In some methods, the treating or preventing results in areduction in plasma glucose in the subject, a reduction in plasmainsulin in the subject, a reduction in body weight and/or food intake,or an increase in glucose tolerance in the subject. In particularembodiments, the glucose metabolism disorder is diabetes mellitus. Insome embodiments, the subject is obese and/or has a body weightdisorder.

Though not limited to any particular route of administration or dosingregimen, in some embodiments the administering is by parenteral (e.g.,subcutaneous) injection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the human GDF15 precursor amino acid sequence and thecorresponding nucleic acid encoding the human GDF15 precursor amino acidsequence.

FIG. 1B depicts the mature human GDF15 amino acid sequence and thecorresponding nucleic acid sequence encoding mature human GDF15.

FIG. 1C depicts a human serum albumin precursor sequence comprisingendogenous signal peptide and prodomain, and mature human serum albumin(D25-L609), and the corresponding nucleic acid sequence; and a humanserum albumin precursor sequence comprising IgK signal peptide andmature human serum albumin (D25-L609), and the corresponding nucleicacid sequence.

FIG. 1D depicts the mature human serum albumin amino acid sequence(subsequence of the amino acid sequence of FIG. 1C lacking the IgKSignal Peptide, and the corresponding nucleic acid sequence).

FIG. 1E depicts a fusion molecule wherein the human serum albumin aminoacid sequence having an IgK signal sequence is fused to the N-terminusof the mature human GDF15 amino acid sequence through aprotease-sensitive 2×(4Gly-Ser) Factor Xa-cleavable linker (SEQ IDNO:56), and the corresponding nucleic acid encoding the fusion molecule.

FIG. 1F depicts a fusion molecule wherein the mature human serum albuminamino acid sequence is fused to the N-terminus of the mature human GDF15amino acid sequence through a protease-sensitive 2×(4Gly-Ser) FactorXa-cleavable linker (SEQ ID NO:56), and the corresponding nucleic acidencoding the fusion molecule.

FIG. 1G depicts a fusion molecule wherein the human serum albumin aminoacid sequence having an IgK signal sequence is fused to the N-terminusof the mature human GDF15 amino acid sequence through a non-cleavable3×(4Gly-Ser) linker (SEQ ID NO:64), and the corresponding nucleic acidencoding the fusion molecule.

FIG. 1H depicts a fusion molecule wherein the mature human serum albuminamino acid sequence is fused to the N-terminus of the mature human GDF15amino acid sequence through a non-cleavable 3×(4Gly-Ser) linker (SEQ IDNO:64), and the corresponding nucleic acid encoding the fusion molecule.

FIGS. 2A-2C depicts the effect on body weight (FIG. 2A), food take (FIG.2B), and blood glucose (FIG. 2C) in ob/ob mice following administrationof the fusion molecule described in FIG. 1H as a single subcutaneousdose at the indicated concentrations (PBS (vehicle), 0.04 mg/kg, 0.12mg/kg, 0.4 mg/kg, and 1.2 mg/kg). As noted in the figure, the indicatedparameters were determined on various days over a 22-day period. In eachgroup of mice, n=7 and p-values (*, p<0.05; **, p<0.01; ***, p<0.001)were determined by student's unpaired T-test comparing the body weight,food intake and blood glucose groups at the various concentrations tovehicle control group at each specified time point.

FIG. 3 depicts the amino acid sequences of the GDF15 muteins generatedvia mutagenesis of predicted solvent-accessible hydrophobic residueswithin mature human GDF15. Fusion molecules were generated wherein eachGDF15 mutein sequence was fused to HSA through the linker depicted inFIG. 1H (a non-cleavable 3×(4Gly-Ser) linker; (SEQ ID NO:64)); thesequences set forth in FIG. 3 neither depict the HSA component nor thelinker component of the fusion molecules.

FIG. 4 is a table summarizing whether each GDF15 mutein set forth inFIG. 3 is secreted as a disulfide-linked homodimer.

FIG. 5 depicts the amino acid sequences of GDF15 muteins having alaninesubstitutions for evaluation of their improvement in physical propertiesrelative to GDF15.

FIG. 6 depicts the amino acid sequences of single-point glycosylationmuteins and additional di-glycosylation muteins for introduction ofN-linked glycosylation consensus sites (Asn-Xxx-Ser/Thr) for evaluationof improved physical properties relative to GDF15. Fusion molecules weregenerated wherein each GDF15 mutein sequence was fused to HSA throughthe linker depicted in FIG. 1E (a Factor Xa-cleavable linker); thesequences set forth in FIG. 6 depict neither the HSA component nor thelinker component of the fusion molecules.

FIG. 7 provides a summary of secretion and dimer formation data, alongwith N-glycan site occupancy, for each engineered N-glycosylated humanGDF15 mutein set forth in FIG. 6.

FIGS. 8A and 8B set forth engineered human GDF15 muteins, followingexpression and purification, having improved physical propertiescompared to mature human GDF15.

FIG. 9A depicts the effect on overnight food intake reduction in ob/obmice following a single, subcutaneous acute dose of 0.3 mg/kg of maturehuman GDF15, N-glycosylated human GDF15 muteins, and vehicle (PBS)control. In each group of mice, n=7 and p-values (*, p<0.05; **, p<0.01;***, p<0.001) were determined by student's unpaired T-test comparingfood intake of GDF15 mutein-treated mice relative to vehicle controlgroup.

FIG. 9B depicts the effect on overnight food intake reduction in DIOmice following a single, subcutaneous acute dose of 1.0 mg/kg of maturehuman GDF15, N-glycosylated human GDF15 muteins, and vehicle (PBS)control. In each group of mice, n=9 and p-values (*, p<0.05; **, p<0.01;***, p<0.001) were determined by student's unpaired T-test comparingfood intake of GDF15 mutein-treated mice relative to vehicle controlgroup.

FIG. 10 indicates that the hydrodynamic radii of GDF15 N-Glycan muteinsare increased relative to mature human GDF15, as determined byanalytical gel filtration chromatography measuring elution time.

FIG. 11A depicts the amino acid sequences of fusion molecules comprisingHSA having an IgK signal sequence fused to the N-terminus of speciesorthologs of mature GDF15 Mus musculus and Macaca mulatta through a2×(4Gly-Ser) Factor Xa-cleavable linker (SEQ ID NO:56).

FIG. 11B depicts the amino acid sequences of fusion molecules comprisingHSA having an IgK signal sequence fused to the N-terminus of maturehuman TGF-β1 and mature human BMP2 through a 2×(4Gly-Ser) FactorXa-cleavable linker (SEQ ID NO:56).

FIG. 12 provides a summary of secretion and dimer formation for fusionmolecules comprising HSA fused to either the N-terminus of speciesorthologs of mature GDF15, or to the N-terminus of mature human TGF-β1or human BMP2 through a cleavable linker.

FIG. 13 provides amino acid sequences of fusion molecules comprising HSAhaving an IgK signal sequence fused to the N-terminus of mature humanGDF15 muteins generated by Alanine-scan through a non-cleavable3×(4Gly-Ser) linker (SEQ ID NO:64).

FIG. 14 provides a summary of secretion and dimer formation for anAlanine scan of GDF15 using fusion molecules comprising HSA amino acidsequence fused to the N-terminus of mature human GDF15 amino acidsequence through a non-cleavable linker. This summary provides atemplate for sites available for mutagenesis that do not impact fold ofGDF15 or secretion parameters.

FIGS. 15A-15E provides multiple sequences of solubility-enhanced,half-life extension molecules comprising fusion to the N-terminus ofmature GDF15 amino acid sequence through various linkers. FIG. 15A):Schematic of fusion molecules comprising a signal sequence fused to Fc(IgG), ABD, and MBD; fused to the N-terminus of mature GDF15 through avariable Linker; FIG. 15B): FcGDF15 containing a 3×(Glu-3Gly-Ser)linker; FIG. 15C): Fc(+)GDF15/Fc(−) charged pair containingFc(+)-3×(Glu-3Gly-Ser)-GDF15 and hFc(−); FIG. 15D): Albumin BindingDomain (ABD) containing a 5×Gly linker (ABD-GDF15); and FIG. 15E):Maltose Binding domain (MBD) containing an Enterokinase cleavable 5×Glylinker (MBD-GDF15).

FIG. 16 provides a summary of (A) the solubility improvements in PBSbuffer of each of the respective fusion constructs described in FIG. 15relative to mature GDF15; and (B) reduction in body weight in ob/obmouse model following a single subcutaneous injection of 3 mg/kg albuminbinding domain fusion to mature GDF15 (ABD-GDF15).

FIG. 17A depicts the amino acid sequence utilized to generaterecombinant platypus (Oa) mature GDF15.

FIG. 17B depicts the effect on overnight food intake and body weightreduction in DIO mice (n=8) following a single, subcutaneous dose of0.001, 0.003, 0.01, 0.03, 0.1, 0.3 and 1.0 mg/kg of mature OaGDF15.

DETAILED DESCRIPTION

Before the methods and compositions of the present disclosure arefurther described, it is to be understood that the disclosure is notlimited to the particular embodiments set forth herein, and it is alsoto be understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “theHuman Polypeptide” includes reference to one or more Human Polypeptides,and so forth. It is further noted that the claims may be drafted toexclude any optional element. As such, this statement is intended toserve as antecedent basis for use of such exclusive terminology such as“solely,” “only” and the like in connection with the recitation of claimelements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Overview

The present disclosure contemplates the use of the agents describedherein, and compositions thereof, to treat and/or prevent variousdiseases, disorders and conditions, and/or the symptoms thereof. In someembodiments, the diseases, disorders and conditions, and/or the symptomsthereof, pertain to glucose metabolism disorders, while in otherembodiments they pertain to body weight disorders. By way of example,but not limitation, the agents, and compositions thereof, can be usedfor the treatment and/or prevention of Type 2 diabetes, insulinresistance and diseases, disorders and conditions characterized byinsulin resistance, decreased insulin production, hyperglycemia,metabolic syndrome, or obesity.

In particular embodiments, the agents contemplated by the presentdisclosure are modified human Growth Differentiation Factor 15 (GDF15),whereas in other embodiments the agents are modified GDF15 variants(e.g., muteins). The modified human GDF15 and modified GDF15 variants(e.g., muteins) have sufficient homology to human GDF15 such that theyhave the ability to bind the GDF15 receptor(s) and initiate a signaltransduction pathway resulting in, for example, reduced body weightand/or the other physiological effects described herein. The presentdisclosure also contemplates nucleic acid molecules encoding theforegoing. As indicated above, the modified human GDF15 molecules andthe modified GDF15 variants described henceforward are collectivelyreferred to as the “Polypeptide(s)”.

Examples of various GDF15 muteins that may be modified are describedhereafter. In some embodiments, one or more GDF15 amino acid residuesare substituted with another amino acid. In other embodiments, one ormore GDF15 native lysine residues are substituted with another aminoacid (however, changes involving K62Q are inactive). In some embodimentsof the present disclosure, alanine scanning may be used to generateGDF15 muteins, and modifications to those muteins can then be assessedfor their ability to enhance one or more desirable properties of themuteins themselves. Examples of modified GDF15 molecules and modifiedGDF15 muteins are described hereafter.

The present disclosure contemplates modifications to GDF15 and GDF15muteins, including, for example, pegylation, glycosylation, and albuminconjugates. In particular embodiments, strategies are employed such thatpegylation is effected only at specific lysine residues (i.e.,site-specific pegylation). In other embodiments, albumin fusions may begenerated whereby mature albumin, or an altered form thereof (e.g., afragment), is conjugated directly or indirectly (e.g., via a linker) toGDF15 or a GDF15 mutein. As indicated above, the modifications may, forexample, improve the serum half-life and/or the solubility of thePolypeptides. Examples of particular modified GDF15 molecules andmodified GDF15 muteins are described hereafter.

DEFINITIONS

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “treat”, “treating”, “treatment” and the like refer to acourse of action (such as administering a Polypeptide or apharmaceutical composition comprising a Polypeptide) initiated after adisease, disorder or condition, or a symptom thereof, has beendiagnosed, observed, and the like so as to eliminate, reduce, suppress,mitigate, or ameliorate, either temporarily or permanently, at least oneof the underlying causes of a disease, disorder, or condition afflictinga subject, or at least one of the symptoms associated with a disease,disorder, condition afflicting a subject. Thus, treatment includesinhibiting (i.e., arresting the development or further development ofthe disease, disorder or condition or clinical symptoms associationtherewith) an active disease (e.g., so as to decrease the level ofinsulin and/or glucose in the bloodstream, to increase glucose toleranceso as to minimize fluctuation of glucose levels, and/or so as to protectagainst diseases caused by disruption of glucose homeostasis).

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering a Polypeptide or apharmaceutical composition comprising a Polypeptide) initiated in amanner (e.g., prior to the onset of a disease, disorder, condition orsymptom thereof) so as to prevent, suppress, inhibit or reduce, eithertemporarily or permanently, a subject's risk of developing a disease,disorder, condition or the like (as determined by, for example, theabsence of clinical symptoms) or delaying the onset thereof, generallyin the context of a subject predisposed to having a particular disease,disorder or condition. In certain instances, the terms also refer toslowing the progression of the disease, disorder or condition orinhibiting progression thereof to a harmful or otherwise undesiredstate.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other caregiver that a subject requires or willbenefit from preventative care. This judgment is made based on a varietyof factors that are in the realm of a physician's or caregiver'sexpertise.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as a part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount that is capable of having any detectable,positive effect on any symptom, aspect, or characteristics of a disease,disorder or condition when administered to a patient. Thetherapeutically effective amount can be ascertained by measuringrelevant physiological effects. For example, in the case of ahyperglycemic condition, a lowering or reduction of blood glucose or animprovement in glucose tolerance test can be used to determine whetherthe amount of an agent is effective to treat the hyperglycemiccondition. For example, a therapeutically effective amount is an amountsufficient to reduce or decrease any level (e.g., a baseline level) offasting plasma glucose (FPG), wherein, for example, the amount issufficient to reduce a FPG level greater than 200 mg/dl to less than 200mg/dl, wherein the amount is sufficient to reduce a FPG level between175 mg/dl and 200 mg/dl to less than the starting level, wherein theamount is sufficient to reduce a FPG level between 150 mg/dl and 175mg/dl to less than the starting level, wherein the amount is sufficientto reduce a FPG level between 125 mg/dl and 150 mg/dl to less than thestarting level, and so on (e.g., reducing FPG levels to less than 125mg/dl, to less than 120 mg/dl, to less than 115 mg/dl, to less than 110mg/dl, etc.). In the case of HbAIc levels, the effective amount is anamount sufficient to reduce or decrease levels by more than about 10% to9%, by more than about 9% to 8%, by more than about 8% to 7%, by morethan about 7% to 6%, by more than about 6% to 5%, and so on. Moreparticularly, a reduction or decrease of HbAIc levels by about 0.1%,0.25%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 3%, 4%, 5%,10%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, or more is contemplated by thepresent disclosure. The therapeutically effective amount can be adjustedin connection with the dosing regimen and diagnostic analysis of thesubject's condition and the like.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., level ofglucose or insulin) or subjective parameter (e.g., a subject's feelingof well-being).

The phrase “glucose tolerance”, as used herein, refers to the ability ofa subject to control the level of plasma glucose and/or plasma insulinwhen glucose intake fluctuates. For example, glucose toleranceencompasses the subject's ability to reduce, within about 120 minutes,the level of plasma glucose back to a level determined before the intakeof glucose.

Broadly speaking, the terms “diabetes” and “diabetic” refer to aprogressive disease of carbohydrate metabolism involving inadequateproduction or utilization of insulin, frequently characterized byhyperglycemia and glycosuria. The terms “pre-diabetes” and“pre-diabetic” refer to a state wherein a subject does not have thecharacteristics, symptoms and the like typically observed in diabetes,but does have characteristics, symptoms and the like that, if leftuntreated, may progress to diabetes. The presence of these conditionsmay be determined using, for example, either the fasting plasma glucose(FPG) test or the oral glucose tolerance test (OGTT). Both usuallyrequire a subject to fast for at least 8 hours prior to initiating thetest. In the FPG test, a subject's blood glucose is measured after theconclusion of the fasting; generally, the subject fasts overnight andthe blood glucose is measured in the morning before the subject eats. Ahealthy subject would generally have a FPG concentration between about90 and about 100 mg/dl, a subject with “pre-diabetes” would generallyhave a FPG concentration between about 100 and about 125 mg/dl, and asubject with “diabetes” would generally have a FPG level above about 126mg/dl. In the OGTT, a subject's blood glucose is measured after fastingand again two hours after drinking a glucose-rich beverage. Two hoursafter consumption of the glucose-rich beverage, a healthy subjectgenerally has a blood glucose concentration below about 140 mg/dl, apre-diabetic subject generally has a blood glucose concentration about140 to about 199 mg/dl, and a diabetic subject generally has a bloodglucose concentration about 200 mg/dl or above. While the aforementionedglycemic values pertain to human subjects, normoglycemia, moderatehyperglycemia and overt hyperglycemia are scaled differently in murinesubjects. A healthy murine subject after a four-hour fast wouldgenerally have a FPG concentration between about 100 and about 150mg/dl, a murine subject with “pre-diabetes” would generally have a FPGconcentration between about 175 and about 250 mg/dl and a murine subjectwith “diabetes” would generally have a FPG concentration above about 250mg/dl.

The term “insulin resistance” as used herein refers to a condition wherea normal amount of insulin is unable to produce a normal physiologicalor molecular response. In some cases, a hyper-physiological amount ofinsulin, either endogenously produced or exogenously administered, isable to overcome the insulin resistance, in whole or in part, andproduce a biologic response.

The term “metabolic syndrome” refers to an associated cluster of traitsthat includes, but is not limited to, hyperinsulinemia, abnormal glucosetolerance, obesity, redistribution of fat to the abdominal or upper bodycompartment, hypertension, dysfibrinolysis, and dyslipidemiacharacterized by high triglycerides, low high density lipoprotein(HDL)-cholesterol, and high small dense low density lipoprotein (LDL)particles. Subjects having metabolic syndrome are at risk fordevelopment of Type 2 diabetes and/or other disorders (e.g.,atherosclerosis).

The phrase “glucose metabolism disorder” encompasses any disordercharacterized by a clinical symptom or a combination of clinicalsymptoms that is associated with an elevated level of glucose and/or anelevated level of insulin in a subject relative to a healthy individual.Elevated levels of glucose and/or insulin may be manifested in thefollowing diseases, disorders and conditions: hyperglycemia, type IIdiabetes, gestational diabetes, type I diabetes, insulin resistance,impaired glucose tolerance, hyperinsulinemia, impaired glucosemetabolism, pre-diabetes, other metabolic disorders (such as metabolicsyndrome, which is also referred to as syndrome X), and obesity, amongothers. The Polypeptides of the present disclosure, and compositionsthereof, can be used, for example, to achieve and/or maintain glucosehomeostasis, e.g., to reduce glucose level in the bloodstream and/or toreduce insulin level to a range found in a healthy subject.

The term “hyperglycemia”, as used herein, refers to a condition in whichan elevated amount of glucose circulates in the blood plasma of asubject relative to a healthy individual. Hyperglycemia can be diagnosedusing methods known in the art, including measurement of fasting bloodglucose levels as described herein.

The term “hyperinsulinemia”, as used herein, refers to a condition inwhich there are elevated levels of circulating insulin when,concomitantly, blood glucose levels are either elevated or normal.Hyperinsulinemia can be caused by insulin resistance which is associatedwith dyslipidemia, such as high triglycerides, high cholesterol, highlow-density lipoprotein (LDL) and low high-density lipoprotein (HDL);high uric acids levels; polycystic ovary syndrome; type II diabetes andobesity. Hyperinsulinemia can be diagnosed as having a plasma insulinlevel higher than about 2 μU/mL.

As used herein, the phrase “body weight disorder” refers to conditionsassociated with excessive body weight and/or enhanced appetite. Variousparameters are used to determine whether a subject is overweightcompared to a reference healthy individual, including the subject's age,height, sex and health status. For example, a subject may be consideredoverweight or obese by assessment of the subject's Body Mass Index(BMI), which is calculated by dividing a subject's weight in kilogramsby the subject's height in meters squared. An adult having a BMI in therange of ˜18.5 to ˜24.9 kg/m² is considered to have a normal weight; anadult having a BMI between ˜25 and ˜29.9 kg/m² may be consideredoverweight (pre-obese); and an adult having a BMI of ˜30 kg/m² or highermay be considered obese. Enhanced appetite frequently contributes toexcessive body weight. There are several conditions associated withenhanced appetite, including, for example, night eating syndrome, whichis characterized by morning anorexia and evening polyphagia oftenassociated with insomnia, but which may be related to injury to thehypothalamus.

The term “Activators” refers to agents that, for example, stimulate,increase, activate, facilitate, enhance activation, sensitize orup-regulate the function or activity of one or more Polypeptides. Inaddition, Activators include agents that operate through the samemechanism of action as the Polypeptides (i.e., agents that modulate thesame signaling pathway as the Polypeptides in a manner analogous to thatof the Polypeptides) and are capable of eliciting a biological responsecomparable to (or greater than) that of the Polypeptides. Examples ofActivators include agonists such as small molecule compounds.

The term “Modulators” collectively refers to the Polypeptides and theActivators.

The terms “modulate”, “modulation” and the like refer to the ability ofan agent (e.g., an Activator) to increase the function or activity ofone or more Polypeptides (or the nucleic acid molecules encoding them),either directly or indirectly; or to the ability of an agent to producean effect comparable to that of one or more Polypeptides.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones. The terms includefusion proteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence, fusion proteins with heterologous andhomologous leader sequences, with or without N-terminus methionineresidues; immunologically tagged proteins; and the like.

It will be appreciated that throughout this disclosure reference is madeto amino acids according to the single letter or three letter codes. Forthe reader's convenience, the single and three letter amino acid codesare provided below:

G Glycine Gly P Proline Pro A Alanine Ala V Valine Val L Leucine Leu IIsoleucine Ile M Methionine Met C Cysteine Cys F Phenylalanine Phe YTyrosine Tyr W Tryptophan Trp H Histidine His K Lysine Lys R ArginineArg Q Glutamine Gln N Asparagine Asn E Glutamic Acid Glu D Aspartic AcidAsp S Serine Ser T Threonine Thr

As used herein, the term “variant” encompasses naturally-occurringvariants (e.g., homologs and allelic variants) andnon-naturally-occurring variants (e.g., muteins). Naturally-occurringvariants include homologs, i.e., nucleic acids and polypeptides thatdiffer in nucleotide or amino acid sequence, respectively, from onespecies to another. Naturally-occurring variants include allelicvariants, i.e., nucleic acids and polypeptides that differ in nucleotideor amino acid sequence, respectively, from one individual to anotherwithin a species. Non-naturally-occurring variants include nucleic acidsand polypeptides that comprise a change in nucleotide or amino acidsequence, respectively, where the change in sequence is artificiallyintroduced, e.g., the change is generated in the laboratory or otherfacility by human intervention (“hand of man”).

The term “native”, in reference to GDF15, refers to biologically active,naturally-occurring GDF15, including biologically active,naturally-occurring GDF15 variants. The term includes the 112 amino acidhuman GDF15 mature sequence.

The term “muteins” as used herein refers broadly to mutated recombinantproteins, i.e., a polypeptide comprising an artificially introducedchange in amino acid sequence, e.g., a change in amino acid sequencegenerated in the laboratory or other facility by human intervention(“hand of man”). These proteins usually carry single or multiple aminoacid substitutions and are frequently derived from cloned genes thathave been subjected to site-directed or random mutagenesis, or fromcompletely synthetic genes. “Muteins” of the present disclosure thusencompass, for example, amino acid substitutions and/or amino aciddeletions (e.g., N-terminal truncations of 1, 2, 3, 4, 5, or 6 or moreamino acids) relative to a reference polypeptide, e.g., relative tomature human GDF15.

As used herein in reference to native human GDF15 or a GDF15 mutein, theterms “modified”, “modification” and the like refer to one or morechanges that enhance a desired property of human GDF15, anaturally-occurring GDF15 variant, or a GDF15 mutein, where the changedoes not alter the primary amino acid sequence of the GDF15.“Modification” includes a covalent chemical modification that does notalter the primary amino acid sequence of the GDF15 polypeptide itself.Such desired properties include, for example, enhancing solubility,prolonging the circulation half-life, increasing the stability, reducingthe clearance, altering the immunogenicity or allergenicity, improvingaspects of manufacturability (e.g., cost and efficiency), and enablingthe raising of particular antibodies (e.g., by introduction of uniqueepitopes) for use in detection assays. Changes to human GDF15, anaturally-occurring GDF15 variant, or a GDF15 mutein that may be carriedout include, but are not limited to, pegylation (covalent attachment ofone or more molecules of polyethylene glycol (PEG), or derivativesthereof); glycosylation (e.g., N-glycosylation), polysialylation andhesylation; maltose binding protein fusion; albumin fusion (e.g., HSAfusion); albumin binding through, for example, a conjugated fatty acidchain (acylation); Fc-fusion; and fusion with a PEG mimetic. Someparticular embodiments entail modifications involving polyethyleneglycol, other particular embodiments entail modifications involvingalbumin, and still other particular modifications entail modificationsinvolving glycosylation.

The terms “DNA”, “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

The term “probe” refers to a fragment of DNA or RNA corresponding to agene or sequence of interest, wherein the fragment has been labeledradioactively (e.g., by incorporating 32^(P) or 35^(S)) or with someother detectable molecule, such as biotin, digoxygenin or fluorescein.As stretches of DNA or RNA with complementary sequences will hybridize,a probe can be used, for example, to label viral plaques, bacterialcolonies or bands on a gel that contain the gene of interest. A probecan be cloned DNA or it can be a synthetic DNA strand; the latter can beused to obtain a cDNA or genomic clone from an isolated protein by, forexample, microsequencing a portion of the protein, deducing the nucleicacid sequence encoding the protein, synthesizing an oligonucleotidecarrying that sequence, radiolabeling the sequence and using it as aprobe to screen a cDNA library or a genomic library.

The term “heterologous” refers to two components that are defined bystructures derived from different sources. For example, in the contextof a polypeptide, a “heterologous” polypeptide may include operablylinked amino acid sequences that are derived from different polypeptides(e.g., a first component comprising a recombinant polypeptide and asecond component derived from a native GDF15 polypeptide). Similarly, inthe context of a polynucleotide encoding a chimeric polypeptide, a“heterologous” polynucleotide may include operably linked nucleic acidsequences that can be derived from different genes (e.g., a firstcomponent from a nucleic acid encoding a polypeptide according to anembodiment disclosed herein and a second component from a nucleic acidencoding a carrier polypeptide). Other exemplary “heterologous” nucleicacids include expression constructs in which a nucleic acid comprising acoding sequence is operably linked to a regulatory element (e.g., apromoter) that is from a genetic origin different from that of thecoding sequence (e.g., to provide for expression in a host cell ofinterest, which may be of different genetic origin than the promoter,the coding sequence or both). For example, a T7 promoter operably linkedto a polynucleotide encoding a GDF15 polypeptide or domain thereof issaid to be a heterologous nucleic acid. In the context of recombinantcells, “heterologous” can refer to the presence of a nucleic acid (orgene product, such as a polypeptide) that is of a different geneticorigin than the host cell in which it is present.

The term “operably linked” refers to linkage between molecules toprovide a desired function. For example, “operably linked” in thecontext of nucleic acids refers to a functional linkage between nucleicacid sequences. By way of example, a nucleic acid expression controlsequence (such as a promoter, signal sequence, or array of transcriptionfactor binding sites) may be operably linked to a second polynucleotide,wherein the expression control sequence affects transcription and/ortranslation of the second polynucleotide. In the context of apolypeptide, “operably linked” refers to a functional linkage betweenamino acid sequences (e.g., different domains) to provide for adescribed activity of the polypeptide.

As used herein in the context of the structure of a polypeptide,“N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxylterminus”) refer to the extreme amino and carboxyl ends of thepolypeptide, respectively, while the terms “N-terminal” and “C-terminal”refer to relative positions in the amino acid sequence of thepolypeptide toward the N-terminus and the C-terminus, respectively, andcan include the residues at the N-terminus and C-terminus, respectively.“Immediately N-terminal” or “immediately C-terminal” refers to aposition of a first amino acid residue relative to a second amino acidresidue where the first and second amino acid residues are covalentlybound to provide a contiguous amino acid sequence.

“Derived from”, in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” aGDF15 polypeptide), is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring GDF15 polypeptide or aGDF15-encoding nucleic acid), and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made. By way ofexample, the term “derived from” includes homologues or variants ofreference amino acid or DNA sequences.

In the context of a polypeptide, the term “isolated” refers to apolypeptide of interest that, if naturally occurring, is in anenvironment different from that in which it may naturally occur.“Isolated” is meant to include polypeptides that are within samples thatare substantially enriched for the polypeptide of interest and/or inwhich the polypeptide of interest is partially or substantiallypurified. Where the polypeptide is not naturally occurring, “isolated”indicates the polypeptide has been separated from an environment inwhich it was made by either synthetic or recombinant means.

“Enriched” means that a sample is non-naturally manipulated (e.g., by ascientist or a clinician) so that a polypeptide of interest is presentin a) a greater concentration (e.g., at least 3-fold greater, at least4-fold greater, at least 8-fold greater, at least 64-fold greater, ormore) than the concentration of the polypeptide in the starting sample,such as a biological sample (e.g., a sample in which the polypeptidenaturally occurs or in which it is present after administration), or b)a concentration greater than the environment in which the polypeptidewas made (e.g., as in a bacterial cell).

“Substantially pure” indicates that a component (e.g., a polypeptide)makes up greater than about 50% of the total content of the composition,and typically greater than about 60% of the total polypeptide content.More typically, “substantially pure” refers to compositions in which atleast 75%, at least 85%, at least 90% or more of the total compositionis the component of interest. In some cases, the polypeptide will makeup greater than about 90%, or greater than about 95% of the totalcontent of the composition.

The terms “antibodies” (Abs) and “immunoglobulins” (Igs) refer toglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific antigen,immunoglobulins include both antibodies and other antibody-likemolecules which lack antigen specificity. Antibodies are described indetail hereafter.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, that is, theindividual antibodies comprising the population are identical except forpossible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. In contrast to polyclonal antibodypreparations, which can include different antibodies directed againstdifferent determinants (epitopes), each monoclonal antibody is directedagainst a single determinant on the antigen.

In the context of an antibody, the term “isolated” refers to an antibodythat has been separated and/or recovered from contaminant components ofits natural environment; such contaminant components include materialswhich might interfere with diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes.

Growth Differentiation Factor 15 (GDF15)

GDF15, also known as MIC-1 (macrophage inhibitory cytokine-1), PDF,PLAB, NAG-1, TGF-PL, and PTGFB, is a member of the transforming growthfactor β (TGF-β) super-family. GDF15, which is synthesized as a 62 kDaintracellular precursor protein that is subsequently cleaved by afurin-like protease, is secreted as a 25 kDa disulfide-linked protein.[See, e.g., Fairlie et al., J. Leukoc. Biol 65:2-5 (1999)]. GDF15 mRNAis seen in several tissues, including liver, kidney, pancreas, colon andplacenta, and GDF15 expression in liver can be significantlyup-regulated during injury of organs such as the liver, kidneys, heartand lungs.

The GDF15 precursor is a 308 amino acid polypeptide (NCBI Ref.Seq.NP_004855.2) containing a 29 amino acid signal peptide, a 167 aminoacid pro-domain, and a mature domain of 112 amino acids which is excisedfrom the pro-domain by furin-like proteases. A 308-amino acid GDF15polypeptide is referred to as a “full-length” GDF15 polypeptide; a112-amino acid GDF15 polypeptide (e.g., amino acids 197-308 of the aminoacid sequence depicted in FIG. 1A) is a “mature” GDF15 polypeptide.Unless otherwise indicated, the term “GDF15” refers to the 112 aminoacid mature sequence. In addition, numerical references to particularGDF15 residues refer to the 112 amino acid mature sequence (i.e.,residue 1 is Ala (A), and residue 112 is Ile (I); see FIG. 1B). Of note,while the GDF15 precursor amino acid sequence predicts three excisionsites, resulting in three putative forms of “mature” human GDF15 (i.e.,110, 112 and 115 amino acids), the 112 amino acid mature sequence isaccepted as being correct.

The scope of the present disclosure includes GDF15 orthologs, andmodified forms thereof, from other mammalian species, and their use,including mouse (NP_035949), chimpanzee (XP_524157), orangutan(XP_002828972), Rhesus monkey (EHH29815), giant panda (XP_002912774),gibbon (XP_003275874), guinea pig (XP_003465238), ferret (AER98997), cow(NP_001193227), pig (NP_001167527), dog (XP_541938) and platypus(Ornithorhynchus anatinus; AFV61279. The mature form of human GDF15 hasapproximately 67% amino acid identity to the mouse ortholog.

A. Modified GDF15 Muteins Having Desired Physical Properties

The present disclosure contemplates, in part, modified GDF15 muteins,wherein one or more amino acid residues of the mature GDF15 polypeptideare substituted with one or more other residues. For example, the GDF15mutein component of a modified GDF15 mutein may include one or moresubstitutions of native lysine residues (i.e., residues 62, 69, 91 and107) with any other amino acid, with the exception that GDF15 muteinscontaining K62Q are inactive. As such, a modified GDF15 containing K62Qmay be specifically excluded from the GDF15 muteins of the presentdisclosure. GDF15 muteins retaining K62 but incorporating anycombination of K69Q, K91R and/or K107R are active in lowering bodyweight to a level comparable to that of mature human GDF15 control.

In other GDF15 muteins, one or more GDF15 residue is substituted withanother amino acid, including, for example, the following substitutions:H18Q, T19S or V20L. Such GDF15 muteins are candidates for modificationto improve one or more inherent physical properties (e.g., stability,serum half-life, and generation of particular antibodies for use indetection assays and protein purification).

Examples of other candidate GDF15 muteins include, but are not limitedto, the following:

-   -   mutein v1) K69Q, K91R, K107R (SEQ ID NO:166);    -   mutein v2) K62Q, K91R, K107R (SEQ ID NO:167);    -   mutein v3) K62Q, K69Q, K107R (SEQ ID NO:168);    -   mutein v4) K62Q, K69Q, K91R (SEQ ID NO:169);    -   mutein v5) K91R, K107R (SEQ ID NO:170);    -   mutein v6) K69Q, K107R (SEQ ID NO:171);    -   mutein v7) K69Q, K91R (SEQ ID NO:172);    -   mutein v8) H18Q, T19S, V20L, K62Q, K69Q, K91R, K107R (SEQ ID        NO:173);    -   mutein v9) H18Q, T19S, V20L, K62Q, K91R, K107R (SEQ ID NO:174);    -   mutein v10) H18Q, T19S, V20L, K62Q, K69Q, K107R (SEQ ID NO:175);        and    -   mutein v11) H18Q, T19S, V20L, K62Q, K69Q, K91R (SEQ ID NO:176).

Further examples of GDF15 muteins include, but are not limited to, ahuman GDF15 polypeptide comprising an amino acid substitution at one,two, three, four or more of R2, N3, G4, D5, H6, P8, L9, G10, P11, G12,R13, R16, L17, H18, T19, V20, R21, S23, L24, E25, D26, L27, G28, W29,D31, W32, V33, L34, S35, R37, E38, V39, Q40, V41, T42, M43, I45, P49,S50, Q51, F52, R53, N56, M57, H58, Q60, I61, T63, S64, H66, R67, L68,K69, P70, D71, T72, V73, P74, P76, V79, P80, S82, N84, P85, M86, V87,L88, I89, Q90, K91, T92, D93, T94, G95, V96, S97, L98, Q99, T100, Y101,D102, D103, L104, L105, K107, D108, H110, and I112, wherein the aminoacid substitution may be a conservative amino acid substitution or anonconservative amino acid substitution. In one example, the GDF15muteins contain an alanine substituted for one, two three, four or moreof R2, N3, G4, D5, H6, P8, L9, G10, P11, G12, R13, R16, L17, H18, T19,V20, R21, S23, L24, E25, D26, L27, G28, W29, D31, W32, V33, L34, S35,R37, E38, V39, Q40, V41, T42, M43, I45, P49, S50, Q51, F52, R53, N56,M57, H58, Q60, I61, T63, S64, H66, R67, L68, K69, P70, D71, T72, V73,P74, P76, V79, P80, S82, N84, P85, M86, V87, L88, I89, Q90, K91, T92,D93, T94, G95, V96, S97, L98, Q99, T100, Y101, D102, D103, L104, L105,K107, D108, H110, and I112. In some embodiments, the GDF15 muteins donot have an amino acid substitution at P36, G46, K62, L65, and/or Y83.In some embodiments, the GDF15 muteins do not have an amino acidsubstitution at C7, C14, C15, C44, C48, C77, C78, C109 and/or C111.

As indicated above and as described in more detail below, native GDF15and GDF15 muteins may be modified through, for example, pegylation(covalent attachment of one or more molecules of polyethylene glycol(PEG), or derivatives thereof); glycosylation (e.g., N-glycosylation);polysialylation; albumin fusion molecules comprising serum albumin(e.g., human serum albumin (HSA), cyno serum albumin, or bovine serumalbumin (BSA)); albumin binding through, for example, a conjugated fattyacid chain (acylation); Fc-fusion; and fusion with a PEG mimetic. Incertain embodiments, the modifications are introduced in a site-specificmanner. In other embodiments, the modifications include a linker.

In particular embodiments, the present disclosure contemplatesmodification of mature human GDF15 and GDF15 muteins by conjugation withalbumin. In other embodiments, the present disclosure contemplatesmodification of mature human GDF15 and GDF15 muteins viaN-glycosylation. The characteristics of albumins and GDF15/GDF15 muteinconjugates thereof (e.g., fusion proteins), and N-glycosylatedGDF15/GDF15 muteins are described further hereafter.

Example 1 indicates the effects on body weight, food intake, and fastedblood glucose of a fusion molecule comprising mature HSA fused to theN-terminus of mature human GDF15 through a non-cleavable 3×(4Gly-Ser)linker (SEQ ID NO:64). Administration of the fusion molecule (whichexhibited improved half-life, expression, secretion and solubilityrelative to unconjugated recombinant human GDF15) resulted insignificant improvement in body weight (FIG. 2A), food intake (FIG. 2B),and non-fasted blood glucose (FIG. 2C) compared to vehicle control.These data demonstrate that an HSA fusion with GDF15 is active, and thatsuch fusion molecules represent a viable approach for enhancing certainbeneficial properties of GDF15 muteins. The data also indicate thatmeasurement of the indicated parameters may be useful as a platform forhigh-throughput screening of muteins.

Example 2 describes the methodology used to identify means for improvingthe physical properties (e.g., solubility and stability) of mature humanGDF15. A set of six hydrophobic residues predicted to besurface-accessible were mutated to alanine as a means of increasingsurface hydrophobicity. Fusion molecules were generated wherein each ofthe six GDF15 mutein sequences was fused to HSA through the linkerdepicted in FIG. 1H (a non-cleavable 3×(4Gly-Ser) linker (SEQ ID NO:64);the sequences set forth in FIG. 3 neither depict the HSA component northe linker component of the fusion molecules.

Thereafter, the fusion molecules were monitored for expression assecreted disulfide-linked homodimers (see FIG. 4). Data generated asdescribed in the examples were used to evaluate solubility resultingfrom introduction of N-linked Glycosylation consensus site(s) along thesequence of mature human GDF15, and to address solubility limitationsassociated with surface hydrophobicities and hydrophilicities inherentto mature human GDF15. The evaluation entailed construction ofGDF15/GDF15 muteins—N-terminal HSA fusion molecules containing a FactorXa proteolytic-sensitive, cleavable linker. Each mutein was generated asa non-HSA fusion using the mature sequence of hGDF15 with an IgK signalpeptide. Reduction of surface hydrophobicity of five GDF15 muteins (w29,w32, w52, w68 and w89; see FIG. 3) was assessed via selectivemutagenesis of hydrophobic residues to alanine and were generated aseither HSA-fusions containing a factor Xa proteolytic-sensitivecleavable linker or as a bacterial refold containing the 112 amino acidsequence of mature hGDF15 muteins containing an N-terminal Methionine.Comparison of the solubility of these five muteins relative to maturehuman GDF15 indicated that w52 and w89 were the only muteins exhibitingimproved solubility.

In addition, the surface hydrophilicity of the following five GDF15mutein sequences (see FIG. 5) was assessed via selective mutagenesis ofacidic residues to alanine: w113, w114, w115, w116 and w117. Comparisonof the relative solubility of these five muteins to mature human GDF15indicated that w116 was the only mutein that exhibited improvedsolubility.

The mature human GDF15 sequence was then assessed for its ability toaccommodate introduction of N-linked Glycosylation consensus site(s). Inthis context, a single amino acid substitution would impart the requiredconsensus site within the mature human GDF15 sequence, the consensussite for N-linked glycosylation being defined as “Asn-Xxx-Ser/Thr”,where “Xxx” cannot be a proline residue. Based on a scan of the maturehuman GDF15 sequence, 14 possible single-point muteins were identifiedthat would accommodate introduction of the N-Glycan consensus site. FIG.6 depicts the sequences of the 14 mono-glycosylation muteins, as well asadditional combinatorial di-Glycosylation muteins. Each of theseengineered N-Glycan muteins was evaluated for both N-glycan siteoccupancy and for secretion as a folded GDF15 homodimer into mammaliantissue culture media. As set forth in FIG. 7, 10 of the 14mono-glycosylated muteins were secreted as folded GDF15 homodimers,whereas 4 (w123, w125, w127 and w129) did not result in dimer formation.Of the 10 mono-glycosylation muteins that secreted as homodimers, two(w121 and w124) exhibited low occupancy and their solubility was notsubsequently evaluated (see FIG. 7).

Engineered human mono-glycosylated GDF15 muteins which were bothsecreted as homodimers and possessed high glycan occupancy within theconsensus site exhibited improved solubility compared to mature humanGDF15 (see Example 3; FIG. 8). These GDF15 muteins were assessed fortheir ability to effect a reduction in food intake, and the data are setforth in FIG. 9A.

Finally, hydrodynamic radii of engineered GDF15 N-Glycan muteinsrelative to mature human GDF15 were assessed utilizing analytical gelfiltration chromatography (see Example 4). As indicated in FIG. 10, eachof the N-linked glycan muteins increased the hydrodynamic radii of thehuman GDF15 disulfide-linked dimer. Thus, each mutein may potentiallyserve as a starting point for generating molecules having, for example,a favorable in vivo half-life.

Relative to mature human GDF15, many of the N-Glycosylation muteinsexhibiting the most substantial improvements in physical properties(e.g., enhancement of solubility and increase in hydrodynamic radii)while maintaining an efficacious food intake reduction, appear to belocalized to a specific epitope/region of human GDF15. Specifically,mutagenesis resulting in the introduction of N-Glycosylation consensussites appeared to be tolerated in the epitope/region spanning Gln90 toLeu98. Thus, though an understanding of preferred regions formutagenesis is not required in order to practice the present disclosure,this epitope/region is believed to be advantageous for introduction ofN-Glycosylation consensus sites.

Human serum albumin (HSA) as a cleavable fusion and expression partnerwas then further exploited for the production and purification ofvarious different GDF15 orthologs and BMP/TGF family members asdescribed in FIGS. 11A, 11B and 12. Based on expression profiling andcharacterization of secreted constructs it appears that human serumalbumin (as per standardized expression template ofIgK-HSA-2×(4Gly-Ser)-IEGR-GDF15 (SEQ ID NO:12)) as a fusion partner iswell suited for the mature human GDF15 amino acid sequence. Theexpression template is optimized for human GDF15 and orthologs whichretain at least 45%, at least 50%, at least 60% at least 65%, 70%, 75%,80%, 85%, 90%, 95% or more sequence identity to the amino acid sequenceof mature human GDF15.

Specific residues that are amenable to amino acid substitution wereidentified by Alanine scanning mutagenesis of the 112 amino acidsequence of mature human GDF15, with the results illustrated in FIGS. 7,8, 9 and 10. The human serum albumin expression fusion was utilized toidentify regions not tolerant of mutagenesis. Cysteine residues were notsubjected to mutation to maintain cysteine knot folding (C7, C14, C15,C44, C48, C77, C78, C109 and C111). Five sites were identified thatwould not tolerate mutagenesis to Alanine: w36, w46, w62, w65 and w83(P36, G46, K62, L65, and Y83, described in FIGS. 13 and 14). Theresultant expression and purification of each Alanine mutein wereassessed for aggregation state and homodimerization fidelity, and as aresult identified residues amenable to mutagenesis without detriment tofold or biological function of GDF15. Examples of residues amenable toamino acid substitution include, but are not limited to: N3A, N3Q, N3E,N3S, N3T, G4P, D5S, D5T, Q40A, Q40E, Q40D, Q40H, M43A, M43V, M43F, Q51A,Q51E, Q51L, Q51H, N56A, N56S, M57A, M571, M57T, Q60A, Q60L, N84A, N84E,N84Q, N84T, M86A, M86V, Q90A, Q90E, Q90E, Q90H, Q95A, Q95E, Q95D, Q95H,Q95T, Q95S.

In particular embodiments, modifications to GDF15 were designed toaddress various undesirable effects, for example, deamidation at residueN3 and/or proteolysis at residue R2. In addition to addressingdeamidation at N3, modifications (e.g., substitutions) may also be madeto other asparagine residues (e.g., N84) within the mature GDF15 inorder to prevent deamidation.

As discussed further in Example 6, having demonstrated that N3 isamenable to mutagenesis without substantial detriment to activity (asillustrated by the N3A mutation described herein), it is reasonable toexpect that other amino acids can be substituted at this site, such asN3Q, N3E, N3T or N3S. The skilled artisan is familiar with techniques(e.g., alignment of mature human GDF15 with a non-human GDF15 ortholog)for identifying amino acid residues that tolerate substitution and aminoacid residues that can be substituted for those in the native sequence.

Other modifications of Polypeptides are contemplated herein. Forexample, deamidation at N3 can create an unnatural Aspartate residue,which can result in an iso-aspartate isomerization due to the presenceof G4 directly C-terminal to the deamidation event (i.e., Asp-Gly site).Deamidation at N3 may be prevented by mutating the isomerization partnerGly to a Pro (G4P) in order to disrupt the Asp-Gly pairing.Additionally, deamidation can be prevented via creation of an N-linkedglycosylation site at N3 via mutagenesis of D5, e.g., to D5T or D5S.

Other embodiments of the present disclosure contemplate elimination ofdeamidation at N3 of mature GDF15 through truncation of residues at theN-terminus. The first 3 residues are removed from the N-terminus in aparticular embodiment, the first 4 residues are removed in a furtherembodiment, the first 5 residues are removed in a sill furtherembodiment, and 6 or more residues are removed in additionalembodiments. Such truncations have the further benefit of correcting forproteolysis at R2 (as the arginine residue at that position has beencleaved off).

The present disclosure contemplates the combination of the modificationsdescribed above (e.g., truncation of amino acid residues at theN-terminus) with one or more other modifications C-terminal to residuethree.

Finally, Example 7 describes engineering of genetic fusions to theN-terminus of mature human GDF15 amino acid sequence. These constructsinclude the design and implementation of fusions containing human Fcdomains, albumin binding domains and maltose binding domains (see FIGS.15 and 16). In each case, the expressed construct was assessed forsecretion fidelity from an appropriate transient expression system, andpurified for assessment of physical properties. In the case of AlbuminBinding Domain (ABD) and Maltose Binding Domain (MBD), a significantincrease to maximum solubility was observed which directly impactsformulation considerations for these molecules as pharmaceutical agents(i.e., maximal formulatable dose). Additionally, FIG. 16B illustratesthe 2-week efficacy (body weight reduction) of an ABD fused to GDF15(ABD-GDF15) following a single acute dose of 3 mg/kg in ob/ob mousemodel. PK profiling of the ABD-GDF15 demonstrated a half-life in excessof 24 hrs in ob/ob mice, demonstrating a desirable improvement of GDF15pharmaceutical properties.

Nucleic acid molecules encoding the Polypeptides are contemplated by thepresent disclosure, including their naturally-occurring andnon-naturally occurring isoforms, allelic variants and splice variants.As previously noted, a Polypeptide also refers to polypeptides that haveone or more alterations in the amino acid residues (e.g., at locationsthat are not conserved across variants or species) while retaining theconserved domains and having the same biological activity as thenaturally-occurring Polypeptides. The presesent disclosure alsoencompasses nucleic acid sequences that vary in one or more bases from anaturally-occurring DNA sequence but still translate into an amino acidsequence that corresponds to a Polypeptide due to degeneracy of thegenetic code. For example, GDF15 may refer to amino acid sequences thatdiffer from the naturally-occurring sequence by one or more conservativesubstitutions, tags, or conjugates (e.g., a Polypeptide).

Thus, in addition to any naturally-occurring GDF15 polypeptide, thepresent disclosure contemplates having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10usually no more than 20, 10, or 5 amino acid substitutions, where thesubstitution is usually a conservative amino acid substitution (e.g., aPolypeptide).

By “conservative amino acid substitution” generally refers tosubstitution of amino acid residues within the following groups: 1) L,I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D,E. Conservative amino acid substitutions preserve the activity of theprotein by replacing an amino acid(s) in the protein with an amino acidwith a side chain of similar acidity, basicity, charge, polarity, orsize of the side chain. Guidance for substitutions, insertions, ordeletions may be based on alignments of amino acid sequences ofdifferent variant proteins or proteins from different species.

The present disclosure also contemplates active fragments (e.g.,subsequences) of the Polypeptides containing contiguous amino acidresidues derived from the mature GDF15 polypeptide or a GDF15 mutein.The length of contiguous amino acid residues of a peptide or apolypeptide subsequence varies depending on the specificnaturally-occurring amino acid sequence from which the subsequence isderived. In general, peptides and polypeptides may be from about 5 aminoacids to about 10 amino acids, from about 10 amino acids to about 15amino acids, from about 15 amino acids to about 20 amino acids, fromabout 20 amino acids to about 25 amino acids, from about 25 amino acidsto about 30 amino acids, from about 30 amino acids to about 40 aminoacids, from about 40 amino acids to about 50 amino acids, from about 50amino acids to about 75 amino acids, from about 75 amino acids to about100 amino acids, or from about 100 amino acids up to the full-lengthpeptide or polypeptide.

Additionally, the Polypeptides can have a defined sequence identitycompared to a reference sequence over a defined length of contiguousamino acids (e.g., a “comparison window”). Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Current Protocols in Molecular Biology(Ausubel et al., eds. 1995 supplement)).

As an example, a suitable Polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, amino acid sequence identity to a contiguous stretch offrom about 5 amino acids to about 10 amino acids, from about 10 aminoacids to about 12 amino acids, from about 12 amino acids to about 15amino acids, from about 15 amino acids to about 20 amino acids, fromabout 20 amino acids to about 25 amino acids, from about 25 amino acidsto about 30 amino acids, from about 30 amino acids to about 35 aminoacids, from about 35 amino acids to about 40 amino acids, from about 40amino acids to about 45 amino acids, from about 45 amino acids to about50 amino acids, from about 50 amino acids to about 60 amino acids, fromabout 60 amino acids to about 70 amino acids, from about 70 amino acidsto about 80 amino acids, from about 80 amino acids to about 90 aminoacids, from about 90 amino acids to about 100 amino acids, or from about100 amino acids to 112 amino acids or 113 amino acids, of one of theamino acid sequence depicted in FIGS. 1, 3, 5, and 6.

The Polypeptides may be isolated from a natural source (e.g., anenvironment other than its naturally-occurring environment) and also maybe recombinantly made (e.g., in a genetically modified host cell such asbacteria; yeast; Pichia; insect cells; and the like), where thegenetically modified host cell is modified with a nucleic acidcomprising a nucleotide sequence encoding the polypeptide. ThePolypeptides may also be synthetically produced (e.g., by cell-freechemical synthesis). Methods of productions are described in more detailbelow.

A Polypeptide may be generated using recombinant techniques tomanipulate different GDF15-related nucleic acids known in the art toprovide constructs capable of encoding the Polypeptide. It will beappreciated that, when provided a particular amino acid sequence, theordinary skilled artisan will recognize a variety of different nucleicacid molecules encoding such amino acid sequence in view of herbackground and experience in, for example, molecular biology.

B. Modulators

The term “Modulators” refers to both Polypeptides and Activators. Asindicated above, Activators are agents that, for example, stimulate,increase, activate, facilitate, enhance activation, sensitize orup-regulate the function or activity of one or more Polypeptides. Inaddition, Activators include agents that operate through the samemechanism of action as the Polypeptides (i.e., agents that modulate thesame signaling pathway as the Polypeptides in a manner analogous to thatof the Polypeptides) and are capable of eliciting a biological responsecomparable to (or greater than) that of the Polypeptides. An Activatormay be, for example, a small molecule agonist compound, or otherbioorganic molecule.

In some embodiments, the Activator is a small molecule agonist compound.Numerous libraries of small molecule compounds (e.g., combinatoriallibraries) are commercially available and can serve as a starting pointfor identifying such an Activator. The skilled artisan is able todevelop one or more assays (e.g., biochemical or cell-based assays) inwhich such compound libraries can be screened in order to identify oneor more compounds having the desired properties; thereafter, the skilledmedicinal chemist is able to optimize such one or more compounds by, forexample, synthesizing and evaluating analogs and derivatives thereof.Synthetic and/or molecular modeling studies can also be utilized in theidentification of an Activator.

In still further embodiments, the Activator is an agonistic polypeptidestructurally distinguishable from the Polypeptides but having comparableactivity. The skilled artisan is able to identify such polypeptideshaving desired properties.

Amide Bond Substitutions

In some cases, a Polypeptide includes one or more linkages other thanpeptide bonds, e.g., at least two adjacent amino acids are joined via alinkage other than an amide bond. For example, in order to reduce oreliminate undesired proteolysis or other means of degradation, and/or toincrease serum stability, and/or to restrict or increase conformationalflexibility, one or more amide bonds within the backbone of aPolypeptide can be substituted.

In another example, one or more amide linkages (—CO—NH—) in aPolypeptide can be replaced with a linkage which is an isostere of anamide linkage, such as —CH₂NH—, CH₂S—, —CH₂CH₂—, —CH═CH-(cis and trans),—COCH₂—, —CH(OH)CH₂— or —CH₂SO—. One or more amide linkages in aPolypeptide can also be replaced by, for example, a reduced isosterepseudopeptide bond. See Couder et al. (1993) Int. J. Peptide ProteinRes. 41:181-184. Such replacements and how to effect are known to thoseof ordinary skill in the art.

Amino Acid Substitutions

One or more amino acid substitutions can be made in a Polypeptide. Thefollowing are non-limiting examples:

a) substitution of alkyl-substituted hydrophobic amino acids, includingalanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyricacid, (S)-cyclohexylalanine or other simple alpha-amino acidssubstituted by an aliphatic side chain from C₁-C₁₀ carbons includingbranched, cyclic and straight chain alkyl, alkenyl or alkynylsubstitutions;

b) substitution of aromatic-substituted hydrophobic amino acids,including phenylalanine, tryptophan, tyrosine, sulfotyrosine,biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,2-benzothienylalanine, 3-benzothienylalanine, histidine, includingamino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro,bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theabove-listed aromatic amino acids, illustrative examples of which are:2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3-or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-, 3′-, or4′-methyl-, 2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine;

c) substitution of amino acids containing basic side chains, includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, including alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀branched, linear, or cyclic) derivatives of the previous amino acids,whether the substituent is on the heteroatoms (such as the alphanitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon,in the pro-R position for example. Compounds that serve as illustrativeexamples include: N-epsilon-isopropyl-lysine,3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine,N,N-gamma, gamma′-diethyl-homoarginine. Included also are compounds suchas alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl groupoccupies the pro-R position of the alpha-carbon. Also included are theamides formed from alkyl, aromatic, heteroaromatic (where theheteroaromatic group has one or more nitrogens, oxygens or sulfur atomssingly or in combination) carboxylic acids or any of the many well-knownactivated derivatives such as acid chlorides, active esters, activeazolides and related derivatives) and lysine, ornithine, or2,3-diaminopropionic acid;

d) substitution of acidic amino acids, including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids;

e) substitution of side chain amide residue, including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine; and

f) substitution of hydroxyl containing amino acids, including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine.

In some cases, a Polypeptide comprises one or more naturally occurringnon-genetically encoded L-amino acids, synthetic L-amino acids orD-enantiomers of an amino acid. For example, a Polypeptide can compriseonly D-amino acids. For example, a Polypeptide can comprise one or moreof the following residues: hydroxyproline, β-alanine, o-aminobenzoicacid, m-aminobenzoic acid, p-aminobenzoic acid, m-aminomethylbenzoicacid, 2,3-diaminopropionic acid, α-aminoisobutyric acid, N-methylglycine(sarcosine), ornithine, citrulline, t-butylalanine, t-butylglycine,N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine,naphthylalanine, pyridylalanine 3-benzothienyl alanine,4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine,4-fluorophenylalanine, penicillamine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-2-thienylalanine,methionine sulfoxide, homoarginine, N-acetyl lysine, 2,4-diamino butyricacid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine,ε-amino hexanoic acid, ω-aminohexanoic acid, ω-aminoheptanoic acid,ω-aminooctanoic acid, ω-aminodecanoic acid, ω-aminotetradecanoic acid,cyclohexylalanine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid,δ-amino valeric acid, and 2,3-diaminobutyric acid.

Additional Modifications

A cysteine residue or a cysteine analog can be introduced into aPolypeptide to provide for linkage to another peptide via a disulfidelinkage or to provide for cyclization of the Polypeptide. Methods ofintroducing a cysteine or cysteine analog are known in the art; see,e.g., U.S. Pat. No. 8,067,532.

A Polypeptide can be cyclized. One or more cysteine or cysteine analogscan be introduced into a Polypeptide, where the introduced cysteine orcysteine analog can form a disulfide bond with a second introducedcysteine or cysteine analog. Other means of cyclization includeintroduction of an oxime linker or a lanthionine linker; see, e.g., U.S.Pat. No. 8,044,175. Any combination of amino acids (or non-amino acidmoiety) that can form a cyclizing bond can be used and/or introduced. Acyclizing bond can be generated with any combination of amino acids (orwith amino acid and —(CH2)_(n)-CO— or —(CH2)_(n)-C₆H₄—CO—) withfunctional groups which allow for the introduction of a bridge. Someexamples are disulfides, disulfide mimetics such as the —(CH2)_(n)-carba bridge, thioacetal, thioether bridges (cystathionine orlanthionine) and bridges containing esters and ethers. In theseexamples, n can be any integer, but is frequently less than ten.

Other modifications include, for example, an N-alkyl (or aryl)substitution (ψ[CONR]), or backbone crosslinking to construct lactamsand other cyclic structures. Other derivatives of the modulatorcompounds of the present disclosure include C-terminal hydroxymethylderivatives, O-modified derivatives (e.g., C-terminal hydroxymethylbenzyl ether), N-terminally modified derivatives including substitutedamides such as alkylamides and hydrazides.

In some cases, one or more L-amino acids in a Polypeptide is replacedwith a D-amino acid.

In some cases, a Polypeptide is a retroinverso analog. Sela and Zisman(1997) FASEB J. 11:449. Retro-inverso peptide analogs are isomers oflinear polypeptides in which the direction of the amino acid sequence isreversed (retro) and the chirality, D- or L-, of one or more amino acidstherein is inverted (inverso) e.g., using D-amino acids rather thanL-amino acids. See, e.g., Jameson et al. (1994) Nature 368:744; andBrady et al. (1994) Nature 368:692.

A Polypeptide can include a “Protein Transduction Domain” (PTD), whichrefers to a polypeptide, polynucleotide, carbohydrate, or organic orinorganic compound that facilitates traversing a lipid bilayer, micelle,cell membrane, organelle membrane, or vesicle membrane. A PTD attachedto another molecule facilitates the molecule traversing a membrane, forexample going from extracellular space to intracellular space, orcytosol to within an organelle. In some embodiments, a PTD is covalentlylinked to the amino terminus of a Polypeptide, while in otherembodiments, a PTD is covalently linked to the carboxyl terminus of aPolypeptide. Exemplary protein transduction domains include, but are notlimited to, a minimal undecapeptide protein transduction domain(corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR;SEQ ID NO:177); a polyarginine sequence comprising a number of argininessufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10,or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer GeneTher. 9(6):489-96); an Drosophila Antennapedia protein transductiondomain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); a truncatedhuman calcitonin peptide (Trehin et al. (2004) Pharm. Research21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci.USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:178); TransportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:179);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:180); and RQIKIWFQNRRMKWKK(SEQ ID NO:181). Exemplary PTDs include, but are not limited to,YGRKKRRQRRR (SEQ ID NO:177), RKKRRQRRR (SEQ ID NO:182); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;Exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following: YGRKKRRQRRR (SEQ ID NO:177); RKKRRQRR (SEQ IDNO:183); YARAAARQARA (SEQ ID NO:184); THRLPRRRRRR (SEQ ID NO:185); andGGRRARRRRRR (SEQ ID NO:186).

The carboxyl group COR₃ of the amino acid at the C-terminal end of aPolypeptide can be present in a free form (R₃=OH) or in the form of aphysiologically-tolerated alkaline or alkaline earth salt such as, e.g.,a sodium, potassium or calcium salt. The carboxyl group can also beesterified with primary, secondary or tertiary alcohols such as, e.g.,methanol, branched or unbranched C₁-C₆-alkyl alcohols, e.g., ethylalcohol or tert-butanol. The carboxyl group can also be amidated withprimary or secondary amines such as ammonia, branched or unbranchedC₁-C₆-alkylamines or C₁-C₆ di-alkylamines, e.g., methylamine ordimethylamine.

The amino group of the amino acid NR₁R₂ at the N-terminus of aPolypeptide can be present in a free form (R₁=H and R₂=H) or in the formof a physiologically-tolerated salt such as, e.g., a chloride oracetate. The amino group can also be acetylated with acids such thatR₁=H and R₂=acetyl, trifluoroacetyl, or adamantyl. The amino group canbe present in a form protected by amino-protecting groups conventionallyused in peptide chemistry such as, e.g., Fmoc, Benzyloxy-carbonyl (Z),Boc, or Alloc. The amino group can be N-alkylated in which R₁ and/orR₂=C₁-C₆ alkyl or C₂-C₈ alkenyl or C₇-C₉ aralkyl. Alkyl residues can bestraight-chained, branched or cyclic (e.g., ethyl, isopropyl andcyclohexyl, respectively).

Particular Modifications to Enhance and/or Mimic GDF15 Function

A Polypeptide can include one or more modifications that enhance aproperty desirable in a protein formulated for therapy (e.g., serumhalf-life), that enable the raising of antibodies for use in detectionassays (e.g., epitope tags), that provide for ease of proteinpurification, etc. Such modifications include, but are not limited to,including pegylation (covalent attachment of one or more molecules ofpolyethylene glycol (PEG), or derivatives thereof); glycosylation (N-and O-linked); polysialylation; albumin fusion; albumin binding througha conjugated fatty acid chain (acylation); Fc-fusion proteins; andfusion with a PEG mimetic.

As set forth herein, the present disclosure contemplates fusionmolecules comprising mature GDF15 polypeptide (e.g., mature human GDF15)or a GDF15 mutein polypeptide (e.g., a mutein of mature human GDF15),wherein the mature GDF15 polypeptide or GDF15 mutein polypeptidecomprises at least one modification that does not alter its amino acidsequence, and wherein the modification improves at least one physicalproperty of the polypeptide or the mutein polypeptide. In oneembodiment, the GDF15 polypeptide or GDF15 mutein polypeptidemodification comprises conjugation with serum albumin (e.g., human serumalbumin (HSA), cyno serum albumin, or bovine serum albumin (BSA)). Insome embodiments, the physical property is solubility.

In embodiments wherein the fusion molecule comprises a modified GDF15polypeptide or a GDF15 mutein polypeptide, either of which is conjugatedto albumin, the solubility of the fusion molecule is improved relativeto unconjugated recombinant human GDF15. In certain embodiments, thefusion molecule has a solubility of at least 1 mg/mL in phosphatebuffered saline (PBS) at pH 7.0, In other embodiments, the fusionmolecule has a solubility of at least 2 mg/mL, at least 3 mg/mL, atleast 4 mg/mL, or at least 5 mg/mL. In other embodiments, the fusionmolecule has a solubility of at least 6 mg/rL in phosphate bufferedsaline (PBS) at pH 7.0, at least 7 mg/mL, at least 8 mg/mL, at least 9mg/mL, or at least 10 mg/mL. In particular embodiments, the fusionmolecule has a solubility of greater than 10 mg/mL.

Pegylation: The clinical effectiveness of protein therapeutics is oftenlimited by short plasma half-life and susceptibility to proteasedegradation. Studies of various therapeutic proteins (e.g., filgrastim)have shown that such difficulties may be overcome by variousmodifications, including conjugating or linking the polypeptide sequenceto any of a variety of nonproteinaceous polymers, e.g., polyethyleneglycol (PEG), polypropylene glycol, or polyoxyalkylenes (see, forexample, typically via a linking moiety covalently bound to both theprotein and the nonproteinaceous polymer, e.g., a PEG). SuchPEG-conjugated biomolecules have been shown to possess clinically usefulproperties, including better physical and thermal stability, protectionagainst susceptibility to enzymatic degradation, increased solubility,longer in vivo circulating half-life and decreased clearance, reducedimmunogenicity and antigenicity, and reduced toxicity.

PEGs suitable for conjugation to a polypeptide sequence are generallysoluble in water at room temperature, and have the general formulaR(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective group such asan alkyl or an alkanol group, and where n is an integer from 1 to 1000.When R is a protective group, it generally has from 1 to 8 carbons. ThePEG conjugated to the polypeptide sequence can be linear or branched.Branched PEG derivatives, “star-PEGs” and multi-armed PEGs arecontemplated by the present disclosure. A molecular weight of the PEGused in the present disclosure is not restricted to any particularrange, but certain embodiments have a molecular weight between 500 and20,000 while other embodiments have a molecular weight between 4,000 and10,000.

The present disclosure also contemplates compositions of conjugateswherein the PEGs have different n values and thus the various differentPEGs are present in specific ratios. For example, some compositionscomprise a mixture of conjugates where n=1, 2, 3 and 4. In somecompositions, the percentage of conjugates where n=1 is 18-25%, thepercentage of conjugates where n=2 is 50-66%, the percentage ofconjugates where n=3 is 12-16%, and the percentage of conjugates wheren=4 is up to 5%. Such compositions can be produced by reactionconditions and purification methods know in the art. For example, cationexchange chromatography may be used to separate conjugates, and afraction is then identified which contains the conjugate having, forexample, the desired number of PEGs attached, purified free fromunmodified protein sequences and from conjugates having other numbers ofPEGs attached.

PEG may be bound to a polypeptide of the present disclosure via aterminal reactive group (a “spacer”). The spacer is, for example, aterminal reactive group which mediates a bond between the free amino orcarboxyl groups of one or more of the polypeptide sequences andpolyethylene glycol. The PEG having the spacer which may be bound to thefree amino group includes N-hydroxysuccinylimide polyethylene glycolwhich may be prepared by activating succinic acid ester of polyethyleneglycol with N-hydroxysuccinylimide. Another activated polyethyleneglycol which may be bound to a free amino group is2,4-bis(0-methoxypolyethyleneglycol)-6-chloro-s-triazine which may beprepared by reacting polyethylene glycol monomethyl ether with cyanuricchloride. The activated polyethylene glycol which is bound to the freecarboxyl group includes polyoxyethylenediamine.

Conjugation of one or more of the polypeptide sequences of the presentdisclosure to PEG having a spacer may be carried out by variousconventional methods. For example, the conjugation reaction can becarried out in solution at a pH of from 5 to 10, at temperature from 4°C. to room temperature, for 30 minutes to 20 hours, utilizing a molarratio of reagent to protein of from 4:1 to 30:1. Reaction conditions maybe selected to direct the reaction towards producing predominantly adesired degree of substitution. In general, low temperature, low pH(e.g., pH=5), and short reaction time tend to decrease the number ofPEGs attached, whereas high temperature, neutral to high pH (e.g.,pH≧7), and longer reaction time tend to increase the number of PEGsattached. Various means known in the art may be used to terminate thereaction. In some embodiments the reaction is terminated by acidifyingthe reaction mixture and freezing at, e.g., −20° C.

The present disclosure also contemplates the use of PEG Mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half-life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the peptide or protein drug ofinterest (e.g., Amunix′ XTEN technology; Mountain View, Calif.). Thisobviates the need for an additional conjugation step during themanufacturing process. Moreover, established molecular biologytechniques enable control of the side chain composition of thepolypeptide chains, allowing optimization of immunogenicity andmanufacturing properties.

Glycosylation:

For purposes of the present disclosure, “glycosylation” is meant tobroadly refer to the enzymatic process that attaches glycans toproteins, lipids or other organic molecules. The use of the term“glycosylation” in conjunction with the present disclosure is generallyintended to mean adding or deleting one or more carbohydrate moieties(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that may or may not be present in the nativesequence. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins involving a change in the natureand proportions of the various carbohydrate moieties present.

Glycosylation can dramatically affect the physical properties ofproteins and can also be important in protein stability, secretion, andsubcellular localization. Indeed, glycosylation of the GDF15- and GDF15mutein-related polypeptides described herein imparts beneficialimprovements to their physical properties. By way of example, but notlimitation, solubility of GDF15/GDF15 muteins can be improved byglycosylation, and such improvement may be substantial (see Examples).The solubility improvement exhibited by such modified GDF15/GDF15muteins can, for example, enable the generation of formulations moresuitable for pharmaceutical administration than non-glycosylatedGDF15/GDF15 muteins. The glycosylated GDF15/GDF15 mutein polypeptidesmay also exhibit enhanced stability. Moreover, the polypeptides mayimprove one or more pharmacokinetic properties, such as half-life.

Proper glycosylation can be essential for biological activity. In fact,some genes from eucaryotic organisms, when expressed in bacteria (e.g.,E. coli) which lack cellular processes for glycosylating proteins, yieldproteins that are recovered with little or no activity by virtue oftheir lack of glycosylation.

Addition of glycosylation sites can be accomplished by altering theamino acid sequence. The alteration to the polypeptide may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues (for O-linked glycosylation sites) or asparagineresidues (for N-linked glycosylation sites). The structures of N-linkedand O-linked oligosaccharides and the sugar residues found in each typemay be different. One type of sugar that is commonly found on both isN-acetylneuraminic acid (hereafter referred to as sialic acid). Sialicacid is usually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycoprotein. A particular embodiment of thepresent disclosure comprises the generation and use of N-glycosylationvariants.

The polypeptide sequences of the present disclosure may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids. Anothermeans of increasing the number of carbohydrate moieties on thepolypeptide is by chemical or enzymatic coupling of glycosides to thepolypeptide. Removal of carbohydrates may be accomplished chemically orenzymatically, or by substitution of codons encoding amino acid residuesthat are glycosylated. Chemical deglycosylation techniques are known,and enzymatic cleavage of carbohydrate moieties on polypeptides can beachieved by the use of a variety of endo- and exo-glycosidases.

Dihydrofolate reductase (DHFR)-deficient Chinese Hamster Ovary (CHO)cells are a commonly used host cell for the production of recombinantglycoproteins. These cells do not express the enzyme beta-galactosidealpha-2,6-sialyltransferase and therefore do not add sialic acid in thealpha-2,6 linkage to N-linked oligosaccharides of glycoproteins producedin these cells.

Polysialylation: The present disclosure also contemplates the use ofpolysialylation, the conjugation of peptides and proteins to thenaturally occurring, biodegradable α-(2→8) linked polysialic acid(“PSA”) in order to improve their stability and in vivopharmacokinetics. PSA is a biodegradable, non-toxic natural polymer thatis highly hydrophilic, giving it a high apparent molecular weight in theblood which increases its serum half-life. In addition, polysialylationof a range of peptide and protein therapeutics has led to markedlyreduced proteolysis, retention of activity in vivo activity, andreduction in immunogenicity and antigenicity (see, e.g., G. Gregoriadiset al., Int. J. Pharmaceutics 300(1-2):125-30). As with modificationswith other conjugates (e.g., PEG), various techniques for site-specificpolysialylation are available (see, e.g., T. Lindhout et al., PNAS108(18)7397-7402 (2011)).

Fusion Proteins. The present disclosure contemplates fusion proteins ofwildtype mature GDF15 (e.g., human GDF15), as well as fusion proteins ofthe Polypeptides of the present disclosure (e.g., modified human GDF15molecules, muteins of human GDF15, modified GDF15 muteins, and thelike). Such fusion proteins are generally comprised of a non-GDF15polypeptide (e.g., albumin (e.g., HSA) or a fragment thereof: ABD; Fcpolypeptide; MBD, which may be referred to herein as a “fusion partner”,conjugated to the wildtype GDF15 polypeptide or Polypeptide of thepresent disclosure at its N-terminus or C-terminus. Optionally, thefusion partner may be conjugated to the wildtype GDF15 or Polypeptidethrough a linker polypeptide. The linker polypeptide may optionally be acleavable linker, e.g., an enzymatically cleavable linker. Examples offusion partners are described below.

Albumin Fusion: Additional suitable components and molecules forconjugation include albumins such as human serum albumin (HSA), cynoserum albumin, and bovine serum albumin (BSA).

Mature HSA (see FIG. 1D), a 585 amino acid polypeptide (˜67 kDa) havinga serum half-life of ˜20 days, is primarily responsible for themaintenance of colloidal osmotic blood pressure, blood pH, and transportand distribution of numerous endogenous and exogenous ligands. Theprotein has three structurally homologous domains (domains I, II andIII), is almost entirely in the alpha-helical conformation, and ishighly stabilized by 17 disulphide bridges. The three primary drugbinding regions of albumin are located on each of the three domainswithin sub-domains IB, IIA and IIIA.

Albumin synthesis takes place in the liver, which produces theshort-lived, primary product preproalbumin. Thus, the full-length HSAhas a signal peptide of 18 amino acids (MKWVTFISLLFLFSSAYS; SEQ IDNO:164) followed by a pro-domain of 6 amino acids (RGVFRR; SEQ II)NO:165); this 24 amino acid residue peptide may be referred to as thepre-pro domain. HSA can be expressed and secreted using its endogenoussignal peptide as a pre-pro-domain (see FIG. 1C). Alternatively, HSA canbe expressed and secreted using a IgK signal peptide (SEQ ID NO:53)fused to a mature construct (D25-L609; of SEQ ID NO:5); in a constructused to generate the experimental data presented herein, the endogenoussignal peptide was replaced with human IgK signal peptide, and theendogenous pro-domain was left out entirely. In turn, preproalbumin israpidly co-translationally cleaved in the endoplasmic reticulum lumen atits amino terminus to produce the stable, 609-amino acid precursorpolypeptide, proalbumin (see FIG. 1C). Proalbumin then passes to theGolgi apparatus, where it is converted to the 585 amino acid maturealbumin by a furin-dependent amino-terminal cleavage. Unless otherwiseindicated, reference herein to “albumin” or to “mature albumin” is meantto refer to HSA.

The primary amino acid sequences, structure, and function of albuminsare highly conserved across species, as are the processes of albuminsynthesis and secretion. Albumin serum proteins comparable to HSA arefound in, for example, cynomolgus monkeys, cows, dogs, rabbits and rats.Of the non-human species, bovine serum albumin (BSA) is the moststructurally similar to HSA. [See, e.g., Kosa et al., J Pharm Sci.96(11):3117-24 (November 2007)]. The present disclosure contemplates theuse of albumin from non-human species, including, but not limited to,those set forth above, in, for example, the drug development process. Incertain embodiments, the non-human species is a cow. In otherembodiments, the non-human species is a cynomolgus monkey.

According to the present disclosure, albumin may be conjugated to a drugmolecule (e.g., a polypeptide described herein) at the carboxylterminus, the amino terminus, both the carboxyl and amino termini, andinternally (see, e.g., U.S. Pat. No. 5,876,969 and U.S. Pat. No.7,056,701). Furthermore, the present disclosure contemplates albuminfusion proteins comprising more than one homologous (e.g., multipleGDF15 mutein molecules) or heterologous (e.g., a GDF15 mutein moleculeand a distinct anti-diabetic agent) drug molecules.

In the HSA-drug molecule conjugates contemplated by the presentdisclosure, various forms of albumin may be used, such as albuminsecretion pre-sequences and variants thereof, fragments and variantsthereof, and HSA variants. Such forms generally possess one or moredesired albumin activities. In additional embodiments, the presentdisclosure involves fusion proteins comprising a polypeptide drugmolecule fused directly or indirectly to albumin, an albumin fragment,and albumin variant, etc., wherein the fusion protein has a higherplasma stability than the unfused drug molecule and/or the fusionprotein retains the therapeutic activity of the unfused drug molecule.In some embodiments, the indirect fusion is effected by a linker, suchas a peptide linker or modified version thereof.

In particular embodiments, the albumin, albumin variant, or albuminfragment is conjugated to a polypeptide comprising the 167 amino acidpro-domain and the 112 amino acid mature domain of the 308 amino acidGDF15 precursor polypeptide; thus, the present disclosure contemplates aGDF15 polypeptide that has a length of from about amino acid residue 30to about amino acid residue 308 of the sequence depicted in FIG. 1A (SEQID NO:1).

The present disclosure contemplates direct expression and production ofthe 112 amino acid mature domain of GDF15 as depicted in FIG. 1B, absentthe 167 amino acid pro-domain, using a signal peptide of appropriatelength to confer secretion from mammalian tissue culture. An example ofa suitable signal peptide to facilitate expression and secretionincludes IgK. As indicated above, the art describes mechanisms by whichother appropriate signal peptides can be identified.

In still other embodiments, albumin serves as an intracellular chaperonfor the expression of a drug molecule. For example, a nucleic acidmolecule (e.g., a vector) encoding a HSA-GDF15/GDF15 mutein fusionprotein may be introduced into a cell. Cellular introduction can be byany means (e.g., transfection or electroporation) known in the art. Theexpressed HSA-GDF15/GDF15 mutein fusion protein may optionally beconjugated through a linker(s). Examples of suitable linkers aredescribed herein. Some embodiments contemplate a peptide linker of, forexample, four-to-six amino acids.

In embodiments wherein the fusion protein comprises a linker, the linkermay be a non-cleavable linker. For example, in one embodiment thepresent disclosure contemplates a fusion molecule wherein the HSAprecursor amino acid sequence is fused to the N-terminus of the maturehuman GDF15 or a GDF15 mutein amino acid sequence through anon-cleavable 3×(4Gly-Ser) linker (SEQ ID NO:64) (see, e.g., FIG. 1G),and in another embodiment the present disclosure contemplates a fusionmolecule wherein the mature HSA amino acid sequence is fused to theN-terminus of the mature human GDF15 or a GDF15 mutein amino acidsequence through a non-cleavable 3×(4Gly-Ser) linker (SEQ ID NO:64)(see, e.g., FIG. 1H).

In other embodiments wherein the fusion protein comprises a linker, thelinker is a cleavable linker. For example, the disclosure contemplates afusion molecule wherein the HSA precursor amino acid sequence is fusedto the N-terminus of the mature human GDF15 or a GDF15 mutein amino acidsequence through a protease-sensitive 2×(4Gly-Ser) Factor Xa-cleavablelinker (SEQ ID NO:56) (see, e.g., FIG. 1E). In other embodiments, thedisclosure contemplates a fusion molecule wherein the mature HSA aminoacid sequence is fused to the N-terminus of the mature human GDF15 or aGDF15 mutein amino acid sequence through a protease-sensitive2×(4Gly-Ser) Factor Xa-cleavable linker (SEQ ID NO:56) (see, e.g., FIG.1F).

Construction of HSA-cleavable linker-mature recombinant GDF15/GDF15mutein molecules, as well as construction of mature recombinantGDF15/GDF15 mutein-cleavable linker-HSA fusion molecules, may be used tofacilitate the assessment of, for example, solubility and thedetermination of in vivo efficacy of the GDF15/GDF15 mutein. In suchembodiments, the GDF15/GDF15 mutein may be excised from the HSAchaperone through intracellular cleavage or through in vitro enzymaticcleavage. In some embodiments, excision is effected by proteolyticdigestion of the cleavable linker using any viable protease. In otherembodiments, GDF15 muteins can also be generated as non-HSA fusions viaconstruction of a signal peptide fused to the mature 112 amino acidsequence, as denoted in FIG. 1B.

Intracellular cleavage may be carried out enzymatically by, for example,furin or caspase. The cells express a low level of these endogenousenzymes, which are capable of cleaving a portion of the fusion moleculesintracellularly; thus, some of the polypeptides are secreted from thecell without being conjugated to HSA, while some of the polypeptides aresecreted in the form of fusion molecules that comprise HSA. Embodimentsof the present disclosure contemplate the use of various furin fusionconstructs. For example, constructs may be designed that comprise thesequence RGRR (SEQ ID NO:222), RKRKKR (SEQ ID NO:223), RKKR (SEQ IDNO:224), or RRRKKR (SEQ ID NO:225). Such constructs can have thefollowing general structure: Igk-HSA(D25-L609 of SEQ IDNO:5)-2×(G4S)-furin sequence-hGDF15(A197-I308 of SEQ ID NO:1).

The present disclosure also contemplates extra-cellular cleavage (i.e.,ex-vivo cleavage) whereby the fusion molecules are secreted from thecell, subjected to purification, then cleaved (e.g., using, for example,a Factor Xa proteolytic-sensitive linker or an enterokinase). It isunderstood that the excision may dissociate the entire HSA-linkercomplex from the mature GDF15 or GDF15 mutein, or less that the entireHSA-linker complex.

As alluded to above, fusion of albumin to one or more polypeptides ofthe present disclosure can, for example, be achieved by geneticmanipulation, such that the DNA coding for HSA, or a fragment thereof,is joined to the DNA coding for the one or more polypeptide sequences.Thereafter, a suitable host can be transformed or transfected with thefused nucleotide sequences in the form of, for example, a suitableplasmid, so as to express a fusion polypeptide. The expression may beeffected in vitro from, for example, prokaryotic or eukaryotic cells, orin vivo from, for example, a transgenic organism. In some embodiments ofthe present disclosure, the expression of the fusion protein isperformed in mammalian cell lines, for example, CHO cell lines.Transformation is used broadly herein to refer to the genetic alterationof a cell resulting from the direct uptake, incorporation and expressionof exogenous genetic material (exogenous DNA) from its surroundings andtaken up through the cell membrane(s). Transformation occurs naturallyin some species of bacteria, but it can also be effected by artificialmeans in other cells.

Furthermore, albumin itself may be modified to extend its circulatinghalf-life. Fusion of the modified albumin to one or more Polypeptidescan be attained by the genetic manipulation techniques described aboveor by chemical conjugation; the resulting fusion molecule has ahalf-life that exceeds that of fusions with non-modified albumin. [SeeWO2011/051489].

Well-established technology platforms exist for the genetic fusion andchemical conjugation of polypeptides (e.g., the Polypeptides describedherein) and recombinant albumin. By way of example, the ALBUFUSE® flexplatform (Novozymes Biopharma A/S; Denmark) can be used to effect thegenetic fusion of one or more recombinant albumin molecules to one ormore Polypeptides, thereby producing a contiguous cDNA encoding thePolypeptide(s) and the albumin(s) to generate a single homogeneousprotein. The platform can be used with, for example, yeast and mammalianhost expression systems. By way of further example, the RECOMBUMIN® Flexplatform (Novozymes Biopharma A/S; Denmark) can be used to effectchemical conjugation of the Polypeptides of the present disclosure torecombinant albumin, without any further derivitization of the albumin.Although conjugation may be performed at several amino acid residues(e.g., lysine and tyrosine), the free thiol at Cys34 is a commonstrategy due to site specificity yielding a more homogenous finalproduct.

Alternative Albumin Binding Strategies: Several albumin —bindingstrategies have been developed as alternatives for direct fusion,including albumin binding through a conjugated fatty acid chain(acylation). Because serum albumin is a transport protein for fattyacids, these natural ligands with albumin-binding activity have beenused for half-life extension of small protein therapeutics. For example,insulin determir (LEVEMIR), an approved product for diabetes, comprisesa myristyl chain conjugated to a genetically-modified insulin, resultingin a long-acting insulin analog.

The present disclosure also contemplates fusion proteins which comprisean albumin binding domain (ABD) polypeptide sequence and the sequence ofone or more of the polypeptides described herein. Any ABD polypeptidesequence described herein or in the literature can be a component of thefusion proteins. The components of the fusion proteins can be optionallycovalently bonded through a linker, such as those linkers describedherein. In some of the embodiments of the present disclosure, the fusionproteins comprise the ABD polypeptide sequence as an N-terminal moietyand the polypeptides described herein as a C-terminal moiety.

The present disclosure also contemplates fusion proteins comprising afragment of an albumin binding polypeptide, which fragment substantiallyretains albumin binding; or a multimer of albumin binding polypeptidesor their fragments comprising at least two albumin binding polypeptidesor their fragments as monomer units.

Without wishing to be bound by any theory, it is believed that thepolypeptides described herein bind to the ABD polypeptide sequence,thereby sequestering the polypeptides in a subject leading to increasedduration of action in the subject.

For a general discussion of ABD and related technologies, see WO2012/050923, WO 2012/050930, WO 2012/004384 and WO 2009/016043.

Fusion Proteins with Maltose Binding Protein or Fragments Thereof: Thepresent disclosure also contemplates fusion proteins which comprise amaltose binding protein (MBP), or fragment thereof, and the amino acidsequence of one or more of the Polypeptides described herein. In someembodiments, the MBP fragment comprises a maltose binding domain (MBD).Any MBP, or fragment thereof, or MBD polypeptide sequence describedherein or known in the art can be a component of the fusion proteins ofthe present disclosure. The components of the fusion proteins can beoptionally covalently bonded through a linker, such as those linkersdescribed herein. In some of the embodiments of the present disclosure,the fusion proteins comprise the MBP, or fragment thereof, or MBDpolypeptide sequence as an N-terminal moiety and the polypeptidesdescribed herein as a C-terminal moiety.

The present disclosure also contemplates fusion proteins comprising afragment of a MBP or MBD polypeptide, which fragment substantiallyretains maltose binding activity; or a multimer of maltose bindingpolypeptides, or fragments thereof (e.g., multimer of a MBD) comprisingat least two maltose binding polypeptides, or fragments thereof, asmonomer units (e.g., two or more MBD polypeptides).

For a general discussion of MBP and MBD and related technologies, see,e.g., Kapust et al. (1999) Protein Sci 8(8):1668-74.

Fc Fusion Proteins.

The present disclosure also contemplates fusion proteins which comprisean Fc polypeptide or fragment thereof, and the amino acid sequence ofone or more of the Polypeptides described herein (e.g., modified humanGDF15 molecules, GDF15 muteins, and modified GDF15 muteins). Any Fcpolypeptide sequence described herein or known in the art can be acomponent of the fusion proteins of the present disclosure. Thecomponents of the fusion proteins can be optionally covalently bondedthrough a linker, such as those linkers described herein. In some of theembodiments of the present disclosure, the fusion proteins comprise theFc polypeptide sequence as an N-terminal moiety and the polypeptidesdescribed herein as a C-terminal moiety.

The present disclosure also contemplates Fc polypeptide fusion partners,and fusion proteins comprising such, where the Fc polypeptide fusionpartner is modified to be one partner of a charged Fc pair. A “partnerof a charged Fc pair” refers to a (i) a “negatively charged” Fc sequence(optionally lacking the hinge region) and comprising a charged pairmutation or (ii) a “positively charged” Fc sequence (optionally lackingthe hinge region) and comprising a charged pair mutation. “Positivelycharged” and “negatively charged” are used herein for ease of referenceto describe the nature of the charge pair mutations in the Fc sequences,and not to indicate that the overall sequence or construct necessarilyhas a positive or negative charge. Charged Fc amino acid sequencessuitable for use in Polypeptide constructs (e.g., GDF15 mutein, modifiedGDF15 muteins) of the present disclosure are described in, for exampleWO 2013/113008.

Examples of a positively charged Fc (“Fc(+)”) include an Fc comprisingan aspartatic acid-to-lysine mutation (E356K) and a glutamicacid-to-lysine mutation (D399K) of an Fc sequence lacking the hingeregion. Examples of a negatively charged Fc (“Fc(−)”) include an Fccomprising two lysine-to-aspartate mutations (K392D, K409D) in an Fcsequence lacking the hinge region. The C-terminal lysine (K477) also mayalso be optionally deleted. When a Fc(+)Polypeptide fusion protein(e.g., Fc(+)GDF mutein fusion protein) and a Fc(−)Polypeptide fusionprotein (e.g., Fc(−)GDF mutein fusion protein) GDF mutein are incubatedtogether, the aspartate residues associate with the lysine residuesthrough electrostatic force, facilitating formation of Fc heterodimersbetween the Fc(+) and the Fc(−) sequences of the Polypeptide fusionproteins.

The present disclosure also contemplates constructs designated “hemi” or“hemiFc” constructs, which comprise two Fc sequences joined in tandem bya linker that connects the N-terminus of a first Fc sequence to theC-terminus of a second Fc sequence. In some embodiments, a monomercomprises a Polypeptide (e.g., a mature modified GDF15 or mutein GDF15)sequence linked to the first Fc sequence by a first linker that connectsthe N-terminus of the GDF15 sequence to the C-terminus of the first Fcsequence, wherein the first Fc sequence is linked to the second Fcsequence by a second linker that connects the N-terminus of the first Fcsequence to the C-terminus of the second Fc sequence. The first andsecond Fc sequences also are associated by the Fc hinge regions. Twosuch monomers associate to form a dimer in which the monomers are linkedvia an interchain disulfide bond between the two Polypeptide sequences.For examples of hemiFc polypeptides suitable for use with the GDFmuteins of the present disclosure see WO 2013/113008.

The present disclosure also contemplates fusion proteins having amultimer of Fc polypeptides, or fragments thereof, including a partnerof a charged Fc pair (e.g., multimer of an Fc).

Conjugation with Other Molecules: Additional suitable components andmolecules for conjugation include, for example, thyroglobulin; tetanustoxoid; Diphtheria toxoid; polyamino acids such aspoly(D-lysine:D-glutamic acid); VP6 polypeptides of rotaviruses;influenza virus hemaglutinin, influenza virus nucleoprotein; KeyholeLimpet Hemocyanin (KLH); and hepatitis B virus core protein and surfaceantigen; or any combination of the foregoing.

Thus, the present disclosure contemplates conjugation of one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another protein (e.g., a protein having anamino acid sequence heterologous to the subject protein), or a carriermolecule. Thus, an exemplary polypeptide sequence can be provided as aconjugate with another component or molecule.

A conjugate modification may result in a polypeptide sequence thatretains activity with an additional or complementary function oractivity of the second molecule. For example, a polypeptide sequence maybe conjugated to a molecule, e.g., to facilitate solubility, storage, invivo or shelf half-life or stability, reduction in immunogenicity,delayed or controlled release in vivo, etc. Other functions oractivities include a conjugate that reduces toxicity relative to anunconjugated polypeptide sequence, a conjugate that targets a type ofcell or organ more efficiently than an unconjugated polypeptidesequence, or a drug to further counter the causes or effects associatedwith a disorder or disease as set forth herein (e.g., diabetes).

A Polypeptide may also be conjugated to large, slowly metabolizedmacromolecules such as proteins; polysaccharides, such as sepharose,agarose, cellulose, cellulose beads; polymeric amino acids such aspolyglutamic acid, polylysine; amino acid copolymers; inactivated virusparticles; inactivated bacterial toxins such as toxoid from diphtheria,tetanus, cholera, leukotoxin molecules; inactivated bacteria; anddendritic cells. Such conjugated forms, if desired, can be used toproduce antibodies against a polypeptide of the present disclosure.

Additional candidate components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes.

Purification methods such as cation exchange chromatography may be usedto separate conjugates by charge difference, which effectively separatesconjugates into their various molecular weights. For example, the cationexchange column can be loaded and then washed with ˜20 mM sodiumacetate, pH˜4, and then eluted with a linear (0 M to 0.5 M) NaClgradient buffered at a pH from about 3 to 5.5, e.g., at pH˜4.5. Thecontent of the fractions obtained by cation exchange chromatography maybe identified by molecular weight using conventional methods, forexample, mass spectroscopy, SDS-PAGE, or other known methods forseparating molecular entities by molecular weight.

Fc-fusion Molecules: In certain embodiments, the amino- orcarboxyl-terminus of a polypeptide sequence of the present disclosurecan be fused with an immunoglobulin Fc region (e.g., human Fc) to form afusion conjugate (or fusion molecule). Fc fusion conjugates have beenshown to increase the systemic half-life of biopharmaceuticals, and thusthe biopharmaceutical product may require less frequent administration.

Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells thatline the blood vessels, and, upon binding, the Fc fusion molecule isprotected from degradation and re-released into the circulation, keepingthe molecule in circulation longer. This Fc binding is believed to bethe mechanism by which endogenous IgG retains its long plasma half-life.More recent Fc-fusion technology links a single copy of abiopharmaceutical to the Fc region of an antibody to optimize thepharmacokinetic and pharmacodynamic properties of the biopharmaceuticalas compared to traditional Fc-fusion conjugates.

Other Modifications: The present disclosure contemplates the use ofother modifications, currently known or developed in the future, of thePolypeptides to improve one or more properties. One such method forprolonging the circulation half-life, increasing the stability, reducingthe clearance, or altering the immunogenicity or allergenicity of apolypeptide of the present disclosure involves modification of thepolypeptide sequences by hesylation, which utilizes hydroxyethyl starchderivatives linked to other molecules in order to modify the molecule'scharacteristics. Various aspects of hesylation are described in, forexample, U.S. Patent Appln. Nos. 2007/0134197 and 2006/0258607.

The present disclosure also contemplates fusion molecules comprisingSUMO as a fusion tag (LifeSensors, Inc.; Malvern, Pa.). Fusion of apolypeptide described herein to SUMO may convey several beneficialeffects on the polypeptide, including enhancement of expression,improvement in solubility, and/or assistance in the development ofpurification methods. SUMO proteases recognize the tertiary structure ofSUMO and cleave the fusion protein at the C-terminus of SUMO, thusreleasing a polypeptide described herein with the desired N-terminalamino acid.

Linkers: Linkers and their use have been described above. Any of theforegoing components and molecules used to modify the polypeptidesequences of the present disclosure may optionally be conjugated via alinker. Suitable linkers include “flexible linkers” which are generallyof sufficient length to permit some movement between the modifiedpolypeptide sequences and the linked components and molecules. Thelinker molecules are generally about 6-50 atoms long. The linkermolecules may also be, for example, aryl acetylene, ethylene glycololigomers containing 2-10 monomer units, diamines, diacids, amino acids,or combinations thereof. Suitable linkers can be readily selected andcan be of any suitable length, such as 1 (e.g., Gly), 2, 3, 4, 5, 6, 7,8, 9, 10, 10-20, 20-30, 30-50 amino acids (e.g., Gly).

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (for example, (GS)_(n), GSGGS_(n) and GGGS_(n),where n is an integer of at least one), glycine-alanine polymers,alanine-serine polymers, and other flexible linkers. Glycine andglycine-serine polymers are relatively unstructured, and therefore mayserve as a neutral tether between components. Exemplary flexible linkersinclude, but are not limited to GGSG (SEQ ID NO:226), GGSGG (SEQ IDNO:227), GSGSG (SEQ ID NO:228), GSGGG (SEQ ID NO:229), GGGSG (SEQ IDNO:230), and GSSSG (SEQ ID NO:231).

In some cases, the linker is a cleavable linker, e.g., an enzymaticallycleavable linker. In other cases, the linker is a non-cleavable linker,e.g., a linker that is not cleaved enzymatically under normalphysiological conditions in vivo.

Examples of suitable linkers include, e.g., (GGGGS)_(n), where n is aninteger from 1 to about 10 (SEQ ID NO:187) (e.g., n=3); GGGSGGGSIEGR(SEQ ID NO:188); GGGGG (SEQ ID NO:189); (EGGGS)_(n), where n is aninteger from 1 to about 10 (SEQ ID NO:190) (e.g., n=3).

For example, a proteolytically cleavable crosslinker can be a matrixmetalloproteinase cleavage site, e.g., a cleavage site for a MMPselected from collagenase-1, -2, and -3 (MMP-1, -8, and -13), gelatinaseA and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11),matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP andMT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy(wherein, X represents an arbitrary residue; Hy, a hydrophobic residue)(SEQ ID NO:191), e.g., Pro-X-X-Hy-(Ser/Thr) (SEQ ID NO:192), e.g.,Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ ID NO:193) or Pro-Leu/Gln-Gly-Met-Thr(SEQ ID NO:194). Another example of a protease cleavage site is aplasminogen activator cleavage site, e.g., a uPA or a tissue plasminogenactivator (tPA) cleavage site. Specific examples of cleavage sequencesof uPA and tPA include sequences comprising Val-Gly-Arg. Another exampleis a thrombin cleavage site, e.g., CGLVPAGSGP (SEQ ID NO:195).Additional suitable linkers comprising protease cleavage sites includelinkers comprising one or more of the following amino acid sequences: 1)SLLKSRMVPNFN (SEQ ID NO:196) or SLLIARRMPNFN (SEQ ID NO:197), cleaved bycathepsin B; SKLVQASASGVN (SEQ ID NO:198) or SSYLKASDAPDN (SEQ IDNO:199), cleaved by an Epstein-Barr virus protease; RPKPQQFFGLMN (SEQ IDNO:200) cleaved by MMP-3 (stromelysin); SLRPLALWRSFN (SEQ ID NO:201)cleaved by MMP-7 (matrilysin); SPQGIAGQRNFN (SEQ ID NO:202) cleaved byMMP-9; DVDERDVRGFASFL (SEQ ID NO:203) cleaved by a thermolysin-like MMP;SLPLGLWAPNFN (SEQ ID NO:204) cleaved by matrix metalloproteinase 2(MMP-2); SLLIFRSWANFN (SEQ ID NO:205) cleaved by cathespin L;SGVVIATVIVIT (SEQ ID NO:206) cleaved by cathepsin D; SLGPQGIWGQFN (SEQID NO:207) cleaved by matrix metalloproteinase 1 (MMP-1); KKSPGRVVGGSV(SEQ ID NO:208) cleaved by urokinase-type plasminogen activator;PQGLLGAPGILG (SEQ ID NO:209) cleaved by membrane type 1matrixmetalloproteinase (MT-MMP); HGPEGLRVGFYESDVMGRGHARLVHVEEPHT (SEQID NO:210) cleaved by stromelysin 3 (or MMP-11), thermolysin, fibroblastcollagenase and stromelysin-1; GPQGLAGQRGIV (SEQ ID NO:211) cleaved bymatrix metalloproteinase 13 (collagenase-3); GGSGQRGRKALE (SEQ IDNO:212) cleaved by tissue-type plasminogen activator (tPA); SLSALLSSDIFN(SEQ ID NO:213) cleaved by human prostate-specific antigen; SLPRFKIIGGFN(SEQ ID NO:214) cleaved by kallikrein (hK3); SLLGIAVPGNFN (SEQ IDNO:215) cleaved by neutrophil elastase; and FFKNIVTPRTPP (SEQ ID NO:216)cleaved by calpain (calcium activated neutral protease).

Methods of Production of Polypeptides

A polypeptide of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis).

A. Chemical Synthesis

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as Fmoc and Boc, areavailable for synthesizing polypeptides of the present disclosure.Details of the chemical synthesis are known in the art (e.g., Ganesan A.2006 Mini Rev. Med. Chem. 6:3-10; and Camarero J. A. et al., 2005Protein Pept Lett. 12:723-8).

Solid phase peptide synthesis may be performed as described hereafter.The α functions (Nα) and any reactive side chains are protected withacid-labile or base-labile groups. The protective groups are stableunder the conditions for linking amide bonds but can be readily cleavedwithout impairing the peptide chain that has formed. Suitable protectivegroups for the α-amino function include, but are not limited to, thefollowing: t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z),o-chlorbenzyloxycarbonyl, bi-phenylisopropyloxycarbonyl,tert-amyloxycarbonyl (Amoc),α,α-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl,2-cyano-t-butoxy-carbonyl, 9-fluorenylmethoxycarbonyl (Fmoc),1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)ethyl (Dde) and the like.

Suitable side chain protective groups include, but are not limited to:acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl),benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom),o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl,2-chlorobenzyl, 2-chlorobenzyloxycarbonyl (2-CIZ), 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl,1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), isopropyl,4-methoxy-2,3-6-trimethylbenzylsulfonyl (Mtr),2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), pivalyl,tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl,trimethylsilyl and trityl (Trt).

In the solid phase synthesis, the C-terminal amino acid is coupled to asuitable support material. Suitable support materials are those whichare inert towards the reagents and reaction conditions for the step-wisecondensation and cleavage reactions of the synthesis process and whichdo not dissolve in the reaction media being used. Examples ofcommercially-available support materials include styrene/divinylbenzenecopolymers which have been modified with reactive groups and/orpolyethylene glycol; chloromethylated styrene/divinylbenzene copolymers;hydroxymethylated or aminomethylated styrene/divinylbenzene copolymersand the like. Polystyrene (1%)-divinylbenzene or TentaGel® derivatizedwith 4-benzyloxybenzyl-alcohol (Wang-anchor) or 2-chlorotrityl chloridecan be used if it is intended to prepare the peptidic acid. In the caseof the peptide amide, polystyrene (1%) divinylbenzene or TentaGel®derivatized with 5-(4′-aminomethyl)-3′,5′-dimethoxyphenoxy)valeric acid(PAL-anchor) or p-(2,4-dimethoxyphenyl-amino methyl)-phenoxy group (Rinkamide anchor) can be used.

The linkage to the polymeric support can be achieved by reacting theC-terminal Fmoc-protected amino acid with the support material with theaddition of an activation reagent in ethanol, acetonitrile,N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran,N-methylpyrrolidone or similar solvents at room temperature or elevatedtemperatures (e.g., between 40° C. and 60° C.) and with reaction timesof, e.g., 2 to 72 hours.

The coupling of the Nα-protected amino acid (e.g., the Fmoc amino acid)to the PAL, Wang or Rink anchor can, for example, be carried out withthe aid of coupling reagents such as N,N′-dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIC) or other carbodiimides,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) or other uronium salts, o-acyl-ureas,benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) or other phosphonium salts, N-hydroxysuccinimides, otherN-hydroxyimides or oximes in the presence or also in the absence of1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, e.g., with theaid of TBTU with addition of HOBt, with or without the addition of abase such as, for example, diisopropylethylamine (DIEA), triethylamineor N-methylmorpholine, e.g., diisopropylethylamine with reaction timesof 2 to 72 hours (e.g., 3 hours in a 1.5 to 3-fold excess of the aminoacid and the coupling reagents, e.g., in a 2-fold excess and attemperatures between about 10° C. and 50° C., e.g., 25° C. in a solventsuch as dimethylformamide, N-methylpyrrolidone or dichloromethane, e.g.,dimethylformamide).

Instead of the coupling reagents, it is also possible to use the activeesters (e.g., pentafluorophenyl, p-nitrophenyl or the like), thesymmetric anhydride of the Nα-Fmoc-amino acid, its acid chloride or acidfluoride under the conditions described above.

The Nα-protected amino acid (e.g., the Fmoc amino acid) can be coupledto the 2-chlorotrityl resin in dichloromethane with the addition of DIEAwith reaction times of 10 to 120 minutes, e.g., 20 minutes, but is notlimited to the use of this solvent and this base.

The successive coupling of the protected amino acids can be carried outaccording to conventional methods in peptide synthesis, typically in anautomated peptide synthesizer. After cleavage of the Nα-Fmoc protectivegroup of the coupled amino acid on the solid phase by treatment with,e.g., piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes,e.g., 2×2 minutes with 50% piperidine in DMF and 1×15 minutes with 20%piperidine in DMF, the next protected amino acid in a 3 to 10-foldexcess, e.g., in a 10-fold excess, is coupled to the previous amino acidin an inert, non-aqueous, polar solvent such as dichloromethane, DMF ormixtures of the two and at temperatures between about 10° C. and 50° C.,e.g., at 25° C. The previously mentioned reagents for coupling the firstNα-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable ascoupling reagents. Active esters of the protected amino acid, orchlorides or fluorides or symmetric anhydrides thereof can also be usedas an alternative.

At the end of the solid phase synthesis, the peptide is cleaved from thesupport material while simultaneously cleaving the side chain protectinggroups. Cleavage can be carried out with trifluoroacetic acid or otherstrongly acidic media with addition of 5%-20% V/V of scavengers such asdimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol,anisole ethanedithiol, phenol or water, e.g., 15% v/vdimethylsulfide/ethanedithiol/m-cresol 1:1:1, within 0.5 to 3 hours,e.g., 2 hours. Peptides with fully protected side chains are obtained bycleaving the 2-chlorotrityl anchor with glacial aceticacid/trifluoroethanol/dichloromethane 2:2:6. The protected peptide canbe purified by chromatography on silica gel. If the peptide is linked tothe solid phase via the Wang anchor and if it is intended to obtain apeptide with a C-terminal alkylamidation, the cleavage can be carriedout by aminolysis with an alkylamine or fluoroalkylamine. The aminolysisis carried out at temperatures between about −10° C. and 50° C. (e.g.,about 25° C.), and reaction times between about 12 and 24 hours (e.g.,about 18 hours). In addition the peptide can be cleaved from the supportby re-esterification, e.g., with methanol.

The acidic solution that is obtained may be admixed with a 3 to 20-foldamount of cold ether or n-hexane, e.g., a 10-fold excess of diethylether, in order to precipitate the peptide and hence to separate thescavengers and cleaved protective groups that remain in the ether. Afurther purification can be carried out by re-precipitating the peptideseveral times from glacial acetic acid. The precipitate that is obtainedcan be taken up in water or tert-butanol or mixtures of the twosolvents, e.g., a 1:1 mixture of tert-butanol/water, and freeze-dried.

The peptide obtained can be purified by various chromatographic methods,including ion exchange over a weakly basic resin in the acetate form;hydrophobic adsorption chromatography on non-derivatizedpolystyrene/divinylbenzene copolymers (e.g., Amberlite® XAD); adsorptionchromatography on silica gel; ion exchange chromatography, e.g., oncarboxymethyl cellulose; distribution chromatography, e.g., on Sephadex®G-25; countercurrent distribution chromatography; or high pressureliquid chromatography (HPLC) e.g., reversed-phase HPLC on octyl oroctadecylsilylsilica (ODS) phases.

B. Recombinant Production

Where a polypeptide is produced using recombinant techniques, thepolypeptide may be produced as an intracellular protein or as a secretedprotein, using any suitable construct and any suitable host cell, whichcan be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E.coli) or a yeast host cell, respectively. Other examples of eukaryoticcells that may be used as host cells include insect cells, mammaliancells, and/or plant cells. Where mammalian host cells are used, they mayinclude human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells(e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos7 and CV1) and hamster cells (e.g., Chinese hamster ovary (CHO) cells).

A variety of host-vector systems suitable for the expression of apolypeptide may be employed according to standard procedures known inthe art. See, e.g., Sambrook et al., 1989 Current Protocols in MolecularBiology Cold Spring Harbor Press, New York; and Ausubel et al. 1995Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods forintroduction of genetic material into host cells include, for example,transformation, electroporation, conjugation, calcium phosphate methodsand the like. The method for transfer can be selected so as to providefor stable expression of the introduced polypeptide-encoding nucleicacid. The polypeptide-encoding nucleic acid can be provided as aninheritable episomal element (e.g., a plasmid) or can be genomicallyintegrated. A variety of appropriate vectors for use in production of apolypeptide of interest are commercially available.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. The expressionvector provides transcriptional and translational regulatory sequences,and may provide for inducible or constitutive expression where thecoding region is operably-linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. In general, the transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7).

Expression constructs generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding proteins of interest. A selectablemarker operative in the expression host may be present to facilitateselection of cells containing the vector. Moreover, the expressionconstruct may include additional elements. For example, the expressionvector may have one or two replication systems, thus allowing it to bemaintained in organisms, for example, in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Inaddition, the expression construct may contain a selectable marker geneto allow the selection of transformed host cells. Selectable genes arewell known in the art and will vary with the host cell used.

Isolation and purification of a protein can be accomplished according tomethods known in the art. For example, a protein can be isolated from alysate of cells genetically modified to express the proteinconstitutively and/or upon induction, or from a synthetic reactionmixture by immunoaffinity purification, which generally involvescontacting the sample with an anti-protein antibody, washing to removenon-specifically bound material, and eluting the specifically boundprotein. The isolated protein can be further purified by dialysis andother methods normally employed in protein purification methods. In oneembodiment, the protein may be isolated using metal chelatechromatography methods. Proteins may contain modifications to facilitateisolation.

The polypeptides may be prepared in substantially pure or isolated form(e.g., free from other polypeptides). The polypeptides can be present ina composition that is enriched for the polypeptide relative to othercomponents that may be present (e.g., other polypeptides or other hostcell components). For example, purified polypeptide may be provided suchthat the polypeptide is present in a composition that is substantiallyfree of other expressed proteins, e.g., less than 90%, less than 60%,less than 50%, less than 40%, less than 30%, less than 20%, less than10%, less than 5%, or less than 1%, of the composition is made up ofother expressed proteins.

Antibodies

The present disclosure provides antibodies, including isolatedantibodies, that specifically bind a GDF15 polypeptide, e.g., a GDF15mutein of the present disclosure. The term “antibody” encompasses intactmonoclonal antibodies, polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies) formed from at least two intactantibodies, and antibody binding fragments including Fab and F(ab)′₂,provided that they exhibit the desired biological activity. The basicwhole antibody structural unit comprises a tetramer, and each tetrameris composed of two identical pairs of polypeptide chains, each pairhaving one “light” chain (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. In contrast, the carboxy-terminal portion of eachchain defines a constant region primarily responsible for effectorfunction. Human light chains are classified as kappa and lambda, whereashuman heavy chains are classified as mu, delta, gamma, alpha, orepsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, andIgE, respectively. Binding fragments are produced by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Binding fragments include Fab, Fab′, F(ab′)₂, Fv, and single-chainantibodies.

Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. The antibody chains all exhibit the samegeneral structure of relatively conserved framework regions (FR) joinedby three hyper-variable regions, also called“complementarity-determining regions” or “CDRs”. The CDRs from the twochains of each pair are aligned by the framework regions, enablingbinding to a specific epitope. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4.

An intact antibody has two binding sites and, except in bifunctional orbispecific antibodies, the two binding sites are the same. A bispecificor bifunctional antibody is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments.

As set forth above, binding fragments may be produced by enzymatic orchemical cleavage of intact antibodies. Digestion of antibodies with theenzyme papain results in two identical antigen-binding fragments, alsoknown as “Fab” fragments, and an “Fc” fragment which has noantigen-binding activity. Digestion of antibodies with the enzyme pepsinresults in a F(ab′)₂ fragment in which the two arms of the antibodymolecule remain linked and comprise two-antigen binding sites. TheF(ab′)₂ fragment has the ability to crosslink antigen.

As used herein, the term “Fab” refers to a fragment of an antibody thatcomprises the constant domain of the light chain and the CH1 domain ofthe heavy chain.

When used herein, the term “Fv” refers to the minimum fragment of anantibody that retains both antigen-recognition and antigen-bindingsites. In a two-chain Fv species, this region includes a dimer of oneheavy-chain and one light-chain variable domain in non-covalentassociation. In a single-chain Fv species, one heavy-chain and onelight-chain variable domain can be covalently linked by a flexiblepeptide linker such that the light and heavy chains can associate in a“dimeric” structure analogous to that in a two-chain Fv species. It isin this configuration that the three CDRs of each variable domaininteract to define an antigen-binding site on the surface of the VH-VLdimer. While the six CDRs, collectively, confer antigen-bindingspecificity to the antibody, even a single variable domain (or half ofan Fv comprising only three CDRs specific for an antigen) has theability to recognize and bind antigen.

When used herein, the term “complementarity determining regions” or“CDRs” refers to parts of immunological receptors that make contact witha specific ligand and determine its specificity.

The term “hypervariable region” refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion generally comprises amino acid residues from a CDR and/or thoseresidues from a “hypervariable loop”.

As used herein, the term “epitope” refers to binding sites forantibodies on protein antigens. Epitopic determinants usually comprisechemically active surface groupings of molecules such as amino acids orsugar side chains, as well as specific three-dimensional structural andcharge characteristics. An antibody is said to bind an antigen when thedissociation constant is ≦1 μM, ≦100 nM, or ≦10 nM. An increasedequilibrium constant (“K_(D)”) means that there is less affinity betweenthe epitope and the antibody, whereas a decreased equilibrium constantmeans that there is more affinity between the epitope and the antibody.An antibody with a K_(D) of “no more than” a certain amount means thatthe antibody will bind to the epitope with the given K_(D) or morestrongly. Whereas K_(D) describes the binding characteristics of anepitope and an antibody, “potency” describes the effectiveness of theantibody itself for a function of the antibody. There is not necessarilya correlation between an equilibrium constant and potency; thus, forexample, a relatively low K_(D) does not automatically mean a highpotency.

The term “selectively binds” in reference to an antibody does not meanthat the antibody only binds to a single substance, but rather that theK_(D) of the antibody to a first substance is less than the K_(D) of theantibody to a second substance. An antibody that exclusively binds to anepitope only binds to that single epitope.

When administered to humans, antibodies that contain rodent (i.e.,murine or rat) variable and/or constant regions are sometimes associatedwith, for example, rapid clearance from the body or the generation of animmune response by the body against the antibody. In order to avoid theutilization of rodent-derived antibodies, fully human antibodies can begenerated through the introduction of human antibody function into arodent so that the rodent produces fully human antibodies. Unlessspecifically identified herein, “human” and “fully human” antibodies canbe used interchangeably. The term “fully human” can be useful whendistinguishing antibodies that are only partially human from those thatare completely, or fully, human. The skilled artisan is aware of variousmethods of generating fully human antibodies.

In order to address possible human anti-mouse antibody responses,chimeric or otherwise humanized antibodies can be utilized. Chimericantibodies have a human constant region and a murine variable region,and, as such, human anti-chimeric antibody responses may be observed insome patients. Therefore, it is advantageous to provide fully humanantibodies against multimeric enzymes in order to avoid possible humananti-mouse antibody or human anti-chimeric antibody responses.

Fully human monoclonal antibodies can be prepared, for example, by thegeneration of hybridoma cell lines by techniques known to the skilledartisan. Other preparation methods involve the use of sequences encodingparticular antibodies for transformation of a suitable mammalian hostcell, such as a CHO cell. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for example,packaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art. Methods for introducing heterologouspolynucleotides into mammalian cells are well known in the art andinclude dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei. Mammalian cell lines available ashosts for expression are well known in the art and include, but are notlimited to, CHO cells, HeLa cells, and human hepatocellular carcinomacells.

The antibodies can be used to detect a Polypeptide of the presentdisclosure. For example, the antibodies can be used as a diagnostic bydetecting the level of one or more Polypeptides of the presentdisclosure in a subject, and either comparing the detected level to astandard control level or to a baseline level in a subject determinedpreviously (e.g., prior to any illness).

Another embodiment of the present disclosure entails the use of one ormore human domain antibodies (dAb). dAbs are the smallest functionalbinding units of human antibodies (IgGs) and have favorable stabilityand solubility characteristics. The technology entails a dAb(s)conjugated to HSA (thereby forming a “AlbudAb”; see, e.g., EP1517921B,WO2005/118642 and WO2006/051288) and a molecule of interest (e.g., apolypeptide sequence of the present disclosure). AlbudAbs are oftensmaller and easier to manufacture in microbial expression systems, suchas bacteria or yeast, than current technologies used for extending theserum half-life of polypeptides. As HSA has a half-life of about threeweeks, the resulting conjugated molecule improves the half-life of themolecule of interest. Use of the dAb technology may also enhance theefficacy of the molecule of interest.

Therapeutic and Prophylactic Uses

The present disclosure provides methods for treating or preventinghyperglycemia, hyperinsulinemia, glucose intolerance, glucose metabolismdisorders, obesity and other body weight disorders, as well as othermetabolic and metabolic-associated diseases, disorders and conditions bythe administration of the Polypeptides, or compositions thereof, asdescribed herein. Such methods may also have an advantageous effect onone or more symptoms associated with a disease, disorder or conditionby, for example, decreasing the severity or the frequency of a symptom.

In order to determine whether a subject may be a candidate for thetreatment or prevention of hyperglycemia, hyperinsulinemia, glucoseintolerance, and/or glucose disorders by the methods provided herein,various diagnostic methods known in the art may be utilized. Suchmethods include those described elsewhere herein (e.g., fasting plasmaglucose (FPG) evaluation and the oral glucose tolerance test (oGTT)).

In order to determine whether a subject may be a candidate for thetreatment or prevention of a body weight disorder (e.g., obesity) by themethods provided herein, parameters such as, but not limited to, theetiology and the extent of the subject's condition (e.g., how overweightthe subject is compared to reference healthy individual) should beevaluated. For example, an adult having a BMI between ˜25 and ˜29.9kg/m² may be considered overweight (pre-obese), while an adult having aBMI of ˜30 kg/m² or higher may be considered obese. For subjects who areoverweight and/or who have poor diets (e.g., diets high in fat andcalories), it is common to initially implement and assess the effect ofmodified dietary habits and/or exercise regimens before initiating acourse of therapy comprising one or more of the Polypeptides of thepresent disclosure. As discussed herein, the Polypeptides can effectappetite suppression.

Pharmaceutical Compositions

The Modulators (e.g., Polypeptides) of the present disclosure may be inthe form of compositions suitable for administration to a subject. Ingeneral, such compositions are “pharmaceutical compositions” comprisingone or more Modulators and one or more pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients. In certainembodiments, the Modulators are present in a therapeutically acceptableamount. The pharmaceutical compositions may be used in the methods ofthe present disclosure; thus, for example, the pharmaceuticalcompositions can be administered ex vivo or in vivo to a subject inorder to practice the therapeutic and prophylactic methods and usesdescribed herein.

The pharmaceutical compositions of the present disclosure can beformulated to be compatible with the intended method or route ofadministration; exemplary routes of administration are set forth herein.Furthermore, the pharmaceutical compositions may be used in combinationwith other therapeutically active agents or compounds (e.g., glucoselowering agents) as described herein in order to treat or prevent thediseases, disorders and conditions as contemplated by the presentdisclosure.

The pharmaceutical compositions typically comprise a therapeuticallyeffective amount of at least one of the Modulators (e.g., Polypeptides)contemplated by the present disclosure and one or more pharmaceuticallyand physiologically acceptable formulation agents. Suitablepharmaceutically acceptable or physiologically acceptable diluents,carriers or excipients include, but are not limited to, antioxidants(e.g., ascorbic acid and sodium bisulfate), preservatives (e.g., benzylalcohol, methyl parabens, ethyl or n-propyl, p-hydroxybenzoate),emulsifying agents, suspending agents, dispersing agents, solvents,fillers, bulking agents, detergents, buffers, vehicles, diluents, and/oradjuvants. For example, a suitable vehicle may be physiological salinesolution or citrate buffered saline, possibly supplemented with othermaterials common in pharmaceutical compositions for parenteraladministration. Neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. Those skilled in the art willreadily recognize a variety of buffers that could be used in thepharmaceutical compositions and dosage forms. Typical buffers include,but are not limited to, pharmaceutically acceptable weak acids, weakbases, or mixtures thereof. As an example, the buffer components can bewater soluble materials such as phosphoric acid, tartaric acids, lacticacid, succinic acid, citric acid, acetic acid, ascorbic acid, asparticacid, glutamic acid, and salts thereof. Acceptable buffering agentsinclude, for example, a Tris buffer,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andN-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

After a pharmaceutical composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form. In some embodiments, the pharmaceutical composition isprovided in a single-use container (e.g., a single-use vial, ampoule,syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas amulti-use container (e.g., a multi-use vial) is provided in otherembodiments. Any drug delivery apparatus may be used to deliver thePolypeptides, including implants (e.g., implantable pumps) and cathetersystems, both of which are well known to the skilled artisan. Depotinjections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the polypeptidesdisclosed herein over a defined period of time. Depot injections areusually either solid- or oil-based and generally comprise at least oneof the formulation components set forth herein. One of ordinary skill inthe art is familiar with possible formulations and uses of depotinjections.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents mentioned herein. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Acceptable diluents,solvents and dispersion media that may be employed include water,Ringer's solution, isotonic sodium chloride solution, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. Moreover, fatty acids such as oleic acid find use in thepreparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, capsules,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups, solutions,microbeads or elixirs. Pharmaceutical compositions intended for oral usemay be prepared according to any method known to the art for themanufacture of pharmaceutical compositions, and such compositions maycontain one or more agents such as, for example, sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tablets,capsules and the like contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets. These excipients may be, for example,diluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

The tablets, capsules and the like suitable for oral administration maybe uncoated or coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate may be employed. They may also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods of preparing liposomes are described in, for example,U.S. Pat. Nos. 4,235,871, 4,501,728, and 4,837,028. Methods for thepreparation of the above-mentioned formulations will be apparent tothose skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients can besuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents, for example a naturally-occurring phosphatide (e.g.,lecithin), or condensation products of an alkylene oxide with fattyacids (e.g., polyoxy-ethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols (e.g., forheptadecaethyleneoxycetanol), or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

The pharmaceutical compositions of the present disclosure may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, forexample, liquid paraffin, or mixtures of these. Suitable emulsifyingagents may be naturally-occurring gums, for example, gum acacia or gumtragacanth; naturally-occurring phosphatides, for example, soy bean,lecithin, and esters or partial esters derived from fatty acids; hexitolanhydrides, for example, sorbitan monooleate; and condensation productsof partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, may be employed.

The present disclosure contemplates the administration of the Modulatorsin the form of suppositories for rectal administration of the drug. Thesuppositories can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include, but are not limited to,cocoa butter and polyethylene glycols.

The Modulators contemplated by the present disclosure may be in the formof any other suitable pharmaceutical composition (e.g., sprays for nasalor inhalation use) currently known or developed in the future.

The concentration of a polypeptide or fragment thereof in a formulationcan vary widely (e.g., from less than about 0.1%, usually at or at leastabout 2% to as much as 20% to 50% or more by weight) and will usually beselected primarily based on fluid volumes, viscosities, andsubject-based factors in accordance with, for example, the particularmode of administration selected.

Routes of Administration

The present disclosure contemplates the administration of the disclosedModulators (e.g., Polypeptides), and compositions thereof, in anyappropriate manner. Suitable routes of administration include parenteral(e.g., intramuscular, intravenous, subcutaneous (e.g., injection orimplant), intraperitoneal, intracisternal, intraarticular,intraperitoneal, intracerebral (intraparenchymal) andintracerebroventricular), oral, nasal, vaginal, sublingual, intraocular,rectal, topical (e.g., transdermal), sublingual and inhalation.

Depot injections, which are generally administered subcutaneously orintramuscularly, may also be utilized to release the Modulatorsdisclosed herein over a defined period of time. Depot injections areusually either solid- or oil-based and generally comprise at least oneof the formulation components set forth herein. One of ordinary skill inthe art is familiar with possible formulations and uses of depotinjections.

Regarding antibodies, in an exemplary embodiment an antibody or antibodyfragment of the present disclosure is stored at 10 mg/ml in sterileisotonic aqueous saline solution for injection at 4° C. and is dilutedin either 100 ml or 200 ml 0.9% sodium chloride for injection prior toadministration to the subject. The antibody is administered byintravenous infusion over the course of 1 hour at a dose of between 0.2and 10 mg/kg. In other embodiments, the antibody is administered byintravenous infusion over a period of between 15 minutes and 2 hours. Instill other embodiments, the administration procedure is viasubcutaneous bolus injection.

Combination Therapy

The present disclosure contemplates the use of the Modulators (e.g.,Polypeptides) in combination with one or more active therapeutic agentsor other prophylactic or therapeutic modalities. In such combinationtherapy, the various active agents frequently have different mechanismsof action. Such combination therapy may be especially advantageous byallowing a dose reduction of one or more of the agents, thereby reducingor eliminating the adverse effects associated with one or more of theagents; furthermore, such combination therapy may have a synergistictherapeutic or prophylactic effect on the underlying disease, disorder,or condition.

As used herein, “combination” is meant to include therapies that can beadministered separately, for example, formulated separately for separateadministration (e.g., as may be provided in a kit), and therapies thatcan be administered together in a single formulation (i.e., a“co-formulation”).

In certain embodiments, the Modulators are administered or appliedsequentially, e.g., where one agent is administered prior to one or moreother agents. In other embodiments, the Modulators are administeredsimultaneously, e.g., where two or more agents are administered at orabout the same time; the two or more agents may be present in two ormore separate formulations or combined into a single formulation (i.e.,a co-formulation). Regardless of whether the two or more agents areadministered sequentially or simultaneously, they are considered to beadministered in combination for purposes of the present disclosure.

The Modulators of the present disclosure can be used in combination withother agents useful in the treatment, prevention, suppression oramelioration of the diseases, disorders or conditions set forth herein,including those that are normally administered to subjects sufferingfrom hyperglycemia, hyperinsulinemia, glucose intolerance, and otherglucose metabolism disorders.

The present disclosure contemplates combination therapy with numerousagents (and classes thereof), including 1) insulin, insulin mimetics andagents that entail stimulation of insulin secretion, includingsulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide,tolbutamide, glyburide, glimepiride, glipizide) and meglitinides (e.g.,repaglinide (PRANDIN) and nateglinide (STARLIX)); 2) biguanides (e.g.,metformin (GLUCOPHAGE)) and other agents that act by promoting glucoseutilization, reducing hepatic glucose production and/or diminishingintestinal glucose output; 3) alpha-glucosidase inhibitors (e.g.,acarbose and miglitol) and other agents that slow down carbohydratedigestion and consequently absorption from the gut and reducepostprandial hyperglycemia; 4) thiazolidinediones (e.g., rosiglitazone(AVANDIA), troglitazone (REZULIN), pioglitazone (ACTOS), glipizide,balaglitazone, rivoglitazone, netoglitazone, troglitazone, englitazone,ciglitazone, adaglitazone, darglitazone that enhance insulin action(e.g., by insulin sensitization), thus promoting glucose utilization inperipheral tissues; 5) glucagon-like-peptides including DPP-IVinhibitors (e.g., vildagliptin (GALVUS) and sitagliptin (JANUVIA)) andGlucagon-Like Peptide-1 (GLP-1) and GLP-1 agonists and analogs (e.g.,exenatide (BYETTA and ITCA 650 (an osmotic pump inserted subcutaneouslythat delivers an exenatide analog over a 12-month period; Intarcia,Boston, Mass.)); 6) and DPP-IV-resistant analogues (incretin mimetics),PPAR gamma agonists, dual-acting PPAR agonists, pan-acting PPARagonists, PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, RXRagonists, glycogen synthase kinase-3 inhibitors, immune modulators,beta-3 adrenergic receptor agonists, 11beta-HSD1 inhibitors, and amylinanalogues.

Furthermore, the present disclosure contemplates combination therapywith agents and methods for promoting weight loss, such as agents thatstimulate metabolism or decrease appetite, and modified diets and/orexercise regimens to promote weight loss.

The Modulators of the present disclosure may be used in combination withone or more other agent in any manner appropriate under thecircumstances. In one embodiment, treatment with the at least one activeagent and at least one Modulator of the present disclosure is maintainedover a period of time. In another embodiment, treatment with the atleast one active agent is reduced or discontinued (e.g., when thesubject is stable), while treatment with the Modulator of the presentdisclosure is maintained at a constant dosing regimen. In a furtherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), while treatment withthe Modulator of the present disclosure is reduced (e.g., lower dose,less frequent dosing or shorter treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), and treatment with theModulator of the present disclosure is increased (e.g., higher dose,more frequent dosing or longer treatment regimen). In yet anotherembodiment, treatment with the at least one active agent is maintainedand treatment with the Modulator of the present disclosure is reduced ordiscontinued (e.g., lower dose, less frequent dosing or shortertreatment regimen). In yet another embodiment, treatment with the atleast one active agent and treatment with the Modulator of the presentdisclosure are reduced or discontinued (e.g., lower dose, less frequentdosing or shorter treatment regimen).

Dosing

The Modulators (e.g., Polypeptides) of the present disclosure may beadministered to a subject in an amount that is dependent upon, forexample, the goal of the administration (e.g., the degree of resolutiondesired); the age, weight, sex, and health and physical condition of thesubject to be treated; the nature of the Modulator, and/or formulationbeing administered; the route of administration; and the nature of thedisease, disorder, condition or symptom thereof (e.g., the severity ofthe dysregulation of glucose/insulin and the stage of the disorder). Thedosing regimen may also take into consideration the existence, nature,and extent of any adverse effects associated with the agent(s) beingadministered. Effective dosage amounts and dosage regimens can readilybe determined from, for example, safety and dose-escalation trials, invivo studies (e.g., animal models), and other methods known to theskilled artisan.

In general, dosing parameters dictate that the dosage amount be lessthan an amount that could be irreversibly toxic to the subject (i.e.,the maximum tolerated dose, “MTD”) and not less than an amount requiredto produce a measurable effect on the subject. Such amounts aredetermined by, for example, the pharmacokinetic and pharmacodynamicparameters associated with absorption, distribution, metabolism, andexcretion (“ADME”), taking into consideration the route ofadministration and other factors.

An effective dose (ED) is the dose or amount of an agent that produces atherapeutic response or desired effect in some fraction of the subjectstaking it. The “median effective dose” or ED50 of an agent is the doseor amount of an agent that produces a therapeutic response or desiredeffect in 50% of the population to which it is administered. Althoughthe ED50 is commonly used as a measure of reasonable expectance of anagent's effect, it is not necessarily the dose that a clinician mightdeem appropriate taking into consideration all relevant factors. Thus,in some situations the effective amount is more than the calculatedED50, in other situations the effective amount is less than thecalculated ED50, and in still other situations the effective amount isthe same as the calculated ED50.

In addition, an effective dose of the Modulators of the presentdisclosure may be an amount that, when administered in one or more dosesto a subject, produces a desired result relative to a healthy subject.For example, an effective dose may be one that, when administered to asubject having elevated plasma glucose and/or plasma insulin, achieves adesired reduction relative to that of a healthy subject by at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, or more than 80%.

An appropriate dosage level will generally be about 0.001 to 100 mg/kgof patient body weight per day, which can be administered in single ormultiple doses. In some embodiments, the dosage level will be about 0.01to about 25 mg/kg per day, and in other embodiments about 0.05 to about10 mg/kg per day. A suitable dosage level may be about 0.01 to 25 mg/kgper day, about 0.05 to 10 mg/kg per day, or about 0.1 to 5 mg/kg perday. Within this range, the dosage may be 0.005 to 0.05, 0.05 to 0.5 or0.5 to 5.0 mg/kg per day.

For administration of an oral agent, the compositions can be provided inthe form of tablets, capsules and the like containing from 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0,15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0,500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the activeingredient. The Modulators may be administered on a regimen of, forexample, 1 to 4 times per day, and often once or twice per day.

The dosage of the Modulators of the present disclosure may be repeatedat an appropriate frequency, which may be in the range of once per dayto once every three months, depending on the pharmacokinetics of theModulators (e.g. half-life) and the pharmacodynamic response (e.g. theduration of the therapeutic effect of the Modulator). In someembodiments where the Modulator is an antibody or a fragment thereof, ora polypeptide or variants thereof, dosing is frequently repeated betweenonce per week and once every 3 months. In other embodiments, suchModulators are administered approximately once per month.

In certain embodiments, the dosage of the disclosed Modulators iscontained in a “unit dosage form”. The phrase “unit dosage form” refersto physically discrete units, each unit containing a predeterminedamount of a Modulator of the present disclosure, either alone or incombination with one or more additional agents, sufficient to producethe desired effect. It will be appreciated that the parameters of a unitdosage form will depend on the particular agent and the effect to beachieved.

Kits

The present disclosure also contemplates kits comprising the disclosedModulators (e.g., Polypeptides), and pharmaceutical compositionsthereof. The kits are generally in the form of a physical structurehousing various components, as described below, and may be utilized, forexample, in practicing the methods described above (e.g., administrationof a Modulator to a subject in need of restoring glucose homeostasis).

A kit can include one or more of the Modulators disclosed herein(provided in, e.g., a sterile container), which may be in the form of apharmaceutical composition suitable for administration to a subject. TheModulators can be provided in a form that is ready for use or in a formrequiring, for example, reconstitution or dilution prior toadministration. When the Modulators are in a form that needs to bereconstituted by a user, the kit may also include buffers,pharmaceutically acceptable excipients, and the like, packaged with orseparately from the Modulators. When combination therapy iscontemplated, the kit may contain the several agents separately or theymay already be combined in the kit. Each component of the kit can beenclosed within an individual container and all of the variouscontainers can be within a single package. A kit of the presentdisclosure can be designed for conditions necessary to properly maintainthe components housed therein (e.g., refrigeration or freezing).

A kit may contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert may be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampoule, tube or vial). Exemplary instructions include thosefor reducing or lowering blood glucose, treatment of hyperglycemia,treatment of diabetes, etc. with the disclosed Modulators, andpharmaceutical compositions thereof.

Labels or inserts can additionally include, or be incorporated into, acomputer readable medium, such as a disk (e.g., hard disk, card, memorydisk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape,or an electrical storage media such as RAM and ROM or hybrids of thesesuch as magnetic/optical storage media, FLASH media or memory-typecards. In some embodiments, the actual instructions are not present inthe kit, but means for obtaining the instructions from a remote source,e.g., via the internet, are provided.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.), but someexperimental errors and deviations should be accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: bp=base pair(s); kb=kilobase(s);pl=picoliter(s); s or sec=second(s); min=minute(s); h or hr=hour(s);aa=amino acid(s); kb=kilobase(s); nt=nucleotide(s); ng=nanogram;μg=microgram; mg=milligram; g=gram; kg=kilogram; dl or dL=deciliter; μlor μL=microliter; ml or mL=milliliter; l or L=liter; μM=micromolar;mM=millimolar; M=molar; kDa=kilodalton; i.m.=intramuscular(ly);i.p.=intraperitoneal(ly); s.c.=subcutaneous(ly); bid=twice daily;HPLC=high performance liquid chromatography; BW=body weight; U=unit;ns=not statistically significant; PG=fasting plasma glucose; FPI=fastingplasma insulin; ITT=insulin tolerance test; PTT=pyruvate tolerance test;oGTT=oral glucose tolerance test; GSIS=glucose-stimulated insulinsecretion; PBS=phosphate-buffered saline; PCR=polymerase chain reaction;NHS=N-Hydroxysuccinimide; DMEM=Dulbeco's Modification of Eagle's Medium;GC=genome copy; EDTA=ethylenediaminetetraacetic acid.

Materials and Methods

The following methods and materials were used in the Examples below:

Animals. Male, 7-15 week-old, B6.V-LEP^(ob)/J (leptin-deficient (ob/ob))mice (The Jackson Laboratory, Bar Harbor, Me.) were used in theexperiments described hereafter. Mice had free access to autoclaveddistilled water and were fed ad libitum a commercial mouse chow(Irradiated 2018 Teklad Global 18% protein Rodent Diet, HarlanLaboratories, Dublin, Va.). Diet-induced obese (DIO) male C57BL/6J mice(The Jackson Laboratory, Bar Harbor, Me.) were maintained on a high-fatdiet (D12492, Research Diets, Inc, New Brunswick, N.J.) containing 60kcal % fat, 20 kcal % protein and 20 kcal % carbohydrate for 12-20weeks. All animal studies were approved by the NGM Institutional AnimalCare and Use Committee.

Nucleic Acid and Amino Acid Sequences. GenBank Accession No. BC000529.2sets forth the cDNA of ORF encoding human GDF15 variants, and GenBankAccession No. NP_004855.2 sets forth the amino acid sequence encoded bythe cDNA. Homo sapiens serum albumin cDNA was purchased from Origene(SC319937), GeneBank Accession No. NM_000477.3, NP_000468).

Fusion PCR fragments for HSA and human GDF15 were generated by Sapphire(Clontech) enzyme, gel purified (Qiagen Gel Extraction kit) andassembled with Gibson Assembly Master Mix (NEB) into pTT5 vectorcontaining the human IgK signal peptide digested with EcoRI and BamHI.Two PCR fragments were generated for cloning, the first encoding HSA andthe second encoding human GDF15. The following primers were used foramplifying HSA: forward primer:5′-tggctccgaggtgccagatgtgatgcacacaagagtgaggttgctcatcgg-3′ (SEQ IDNO:217); reverse primer: 5′-gctaccgcctccacctaagcctaaggcagcttgacttgc-3′(SEQ ID NO:218). The following primers were used for amplifying GDF15:forward primer:5′-gctgccttaggcttaggtggaggcggtagcggtggaggtgggagtggaggtggaggcagtgcgcgcaacggggaccactgtccgctcggg-3′(SEQ ID NO:219); reverse primer:5′-cagaggtcgaggtcgggggatcctcatatgcagtggcagtctttggctaacaa-3′ (SEQ IDNO:220).

Colonies were plated and sequence confirmed. Primers were designed tomutate regions of interest, and mutagenesis was performed withQuikchange Lightning Site Directed Mutagenesis Kit (Agilent).Sequence-confirmed colonies were amplified and plasmid DNA was purifiedusing Qiagen DNA-Maxi prep kit.

HSA-GDF15 Mutein Fusion Molecule Expression. All muteins weretransiently transfected in Expi 293F cells (Invitrogen Corporation,Carlsbad, Calif.). Cells were routinely subcultured in Expi expressionmedium (Invitrogen) and maintained as suspension cultures in shakeflasks of varying sizes. Typically, cells were subcultured at a celldensity of 5e5 viable cells/ml and grown for 3 days before subculturing.The flasks were maintained in a humidified CO₂ incubator (37° C. and 5%CO₂) on New Brunswick shaker platforms (New Brunswick ScientificCompany, Edison, N.J.) at an agitation rate of 110 RPM.

Transfections were performed when the cell density of the culturereached 2.5e6 viable cells/mL at greater than 95% viability. Typically,for 50 mL transfection, 2.5e6 cells/mL×50 mL cells were inoculated in a250 mL shaker flask in 42.5 mL culture volume. Fifty micrograms (50 μg)plasmid DNA consisting of the expression vector containing the gene ofinterest was first diluted in 2.5 mL OPTI-MEM reduced-serum medium(Invitrogen). Simultaneously, Expifectamine transfection reagent(Invitrogen), 2.67 times the volume (of the amount of plasmid DNA) wasalso diluted in 2.5 mL OPTI-MEM reduced-serum medium. After a 5 minincubation at room temperature, the diluted transfection reagent wasslowly added to the diluted plasmid DNA to form transfection competentcomplexes. After a further 20 min incubation period at room temperature,5 mL of the transfection complex was added to the 42.5 mL cell culture.The transfected cells were then placed in the humidified CO₂ incubatoron an orbital shaker maintained at 110 RPM. Twenty-four hourspost-transfection, the transfected culture was fed with 250 μL enhancer1 solution (Invitrogen) and 2.5 mL enhancer 2 solution (Invitrogen). Theculture was then replaced in the humidified CO₂ incubator on an orbitalshaker. Six-to-seven days post-transfection, cultures were harvested bycentrifugation at 3000 RPM for 30 min before being filtered through a0.2 μm filter (Nalgene). Samples were then analyzed on a commassie staingel for expression.

Cleavage of HSA-GDF15 Fusion Molecules. Human Serum Albumin hGDF15fusion constructs were purified to greater than 95% homogeneity. Muteinswere excised via overnight digestion at Room Temperature using Factor Xaobtained from New England Biolabs (P8010) using a 1:500 (w/w) addition(in 1× phosphate buffered saline). Following cleavage, GDF15 muteinswere purified to greater than 95% homogeneity.

Production of Mature GDF15 Fusion Molecules. hGDF15 muteins were alsoconstructed and purified as non-HSA fusions via utilization of a IgKsignal peptide fused to the mature (112 amino acid) sequence of GDF15(FIG. 1B). Mature muteins were purified directly from media to greaterthan 95% homogeneity.

Production of Bacterially-refolded Mature, Non-glycosylated Molecules.hGDF15 muteins set forth in FIG. 4 (w29, w32, w52, w68 and w89) werealso generated as bacterial refolds from inclusion bodies and purifiedto greater than 95% homogeneity containing the mature (112 amino acid)sequence of GDF15 (FIG. 1B) with an N-terminal methionine.

Solubility Assessment of Human GDF15 Muteins. Solubility of GDF15muteins was assessed using two complementary assays, the results ofwhich closely mirrored each other. In one assay format, muteins weredialyzed into 1× phosphate buffered saline and concentrated using AmiconUltra Centrifugal Filters composed of Regenerated Nitrocellulose 10,000NMWL (UFC901096). Following concentration, solubility assessments wereperformed on a NanoDrop ND-1000 spectrophotometer blanked in 1×Phosphate Buffered Saline using Absorbance at 280 nm wavelength andBeer's law (Extinction coefficient=14400, Molecular weight=12,287 Da).In a second assay format, muteins were dialyzed into 0.05% (v/v) formicacid (pH 2.0) and concentrated using Amicon Ultra Centrifugal Filterscomposed of Regenerated Nitrocellulose 10,000 NMWL (UFC901096) up to andgreater than 20 mg/mL. The starting concentration for each mutein wasdetermined using Absorbance at 280 nm wavelength and Beer's law(Extinction coefficient=14400, Molecular weight=12,287 Da). Each muteinwas then serial diluted 2-fold back into 0.01% formic acid and 90 μL ofeach dilution was added to a 96-well plate. 10 μL of 10×PBS was added toeach well and pH was confirmed to be 7.3. Following incubation at roomtemperature overnight with shaking, turbidity was measured at 370 nm.The inflection point at which turbidity begins to occur is accepted asthe maximum solubility for each mutein.

Blood Glucose Assay. Blood glucose from mouse tail snip was measuredusing ACCU-CHEK Active test strips read by ACCU-CHEK Active meter (RocheDiagnostics, Indianapolis, Ind.) following manufacturer's instruction.

Serum GDF15 Muteins Exposure Level Assay. Whole blood (˜50 μl/mouse)from mouse tail snips was collected into plain capillary tubes (BD ClayAdams SurePrep, Becton Dickenson, Sparks, Md.). Serum and blood cellswere separated by spinning the tubes in an Autocrit Ultra 3 (BectonDickinson, Sparks, Md.). GDF15 exposure levels in serum were determinedusing Human GDF-15 Quantikine ELISA Kit (R&D Systems, Minneapolis,Minn.) by following the manufacturer's instructions.

Analytical Gel Filtration. Increases to hydrodynamic radii of engineeredhuman GDF15 muteins was monitored via A280 elution absorbance on anAgilent 1200-series HPLC elution times (min) using a TOSOH BiosciencesTSKgelG3000SW_(xL) column (7.8 mm ID×30 cm, 5 μm), pre-equilibrated with1× phosphate buffered saline with flow rate of 1 mL/min.

Production of Mature Platypus (Oa) GDF15 Molecule. Referring to FIG.17A, mature OaGDF15 was constructed from a precursor amino acid sequencecontaining a signal sequence, pro-domain (furin cut site underlined),followed by MatureOaGDF15 (bold) (see FIG. 17A).

The OaGDF15 construct was transiently transfected in Expi 293F cells(Invitrogen; Carlsbad, Calif.) in a manner as described herein forHSA-GDF15 mutein fusion molecules. Secreted OaGDF15 was purified togreater than 95% homogeneity from the cell media. The N-terminus of themature form of OaGDF15 was confirmed via LC/MS analysis.

Example 1 Effects of a HSA-GDF15 Fusion Molecule on Body Weight, FoodIntake and Fasted Blood

The effects of a subcutaneously administered fusion molecule havingrecombinant HSA fused to recombinant human GDF15 on body weight, foodintake, and fasted blood glucose were evaluated over a 22 day periodpost-delivery. Briefly, the fusion molecule depicted in FIG. 1H (matureHSA fused to the N-terminus of mature human GDF15 through anon-cleavable 3×(4Gly-Ser) linker (SEQ ID NO:64) was administered, atvarious doses, as a single, subcutaneous bolus injection to 7-15 weekold male ob/ob mice weighing approximately 44 g and having non-fastedglucose serum levels of approximately 340 mg/dl. Followingadministration of vehicle control (PBS) or the fusion molecule at dosesof 0.04 mg/kg, 0.12 mg/kg, 0.4 mg/kg and 1.2 mg/kg, the indicatedparameters were determined over a 22-day period on days 0, 2, 3, 6, 8,15 and 22. Serum exposure was monitored by Human GDF-15 Quantikine ELISAKit (R&D Systems, Minneapolis, Minn.) following the manufacturer'sinstructions.

The fusion molecule demonstrated an improved half-life of 37 hourscompared to a half-life of 2 hours for unconjugated, recombinant humanGDF15. Additionally, whereas the solubility of human GDF15 is less than0.2 mg/mL in vehicle control buffer (1×PBS), the fusion moleculeimproved solubility to more than 50 mg/mL in vehicle control buffer(1×PBS).

As depicted in FIG. 2, administration of the fusion molecule at doses of0.04 mg/kg, 0.12 mg/kg, 0.4 mg/kg and 1.2 mg/kg resulted in significantimprovement in body weight (FIG. 2A), food intake (FIG. 2B), andnon-fasted blood glucose (FIG. 2C) compared to vehicle control. In eachgroup of mice, n=7 and p-values (*, p<0.05; **, p<0.01; ***, p<0.001)were determined by student's unpaired T-test comparing the body weight,food intake and blood glucose groups at the various concentrations tovehicle control group at each specified time point.

Referring to FIG. 2A, 22 days post-administration of the fusion moleculeat the indicated doses compared to 22 days post-administration ofvehicle control (PBS-injected ob/ob mice (52.5 g)) resulted in thefollowing body weight reductions: a decrease of 1.7 g comprising apercent decrease of 3.2% (ns) for the 0.04 mg/kg dose group; a decreaseof 1.8 g comprising a percent decrease of 3.5% (ns) for the 0.12 mg/kgdose group; a decrease of 1.9 g comprising a percent decrease of 3.6%(*, p<0.05) for the 0.40 mg/kg dose group; and a decrease of 3.2 gcomprising a percent decrease of 6.1% (**, p<0.01) for the 1.2 mg/kgdose group.

Food intake (grams/animal/day) in ob/ob mice administered vehiclecontrol or the fusion molecule at the indicated doses was assessed atvarious times during the 22 day post-administration observation period.Referring to the 9-15 day time period in FIG. 2B, average food intakerelative to vehicle control (PBS)-injected ob/ob mice (7.88g/animal/day) was as follows: average food intake decreased 0.24g/animal/day, which comprised a percent decrease of 3.0% (ns) for 0.04mg/kg dose group; decreased 0.92 g/animal/day, which comprised a percentdecrease of 11.7% (ns) for 0.12 mg/kg dose group; decreased 1.70g/animal/day, which comprised a percent decrease of 21.5% (**, p<0.01)for 0.40 mg/kg dose group; and decreased 2.31 g/animal/day, whichcomprised a percent decrease of 29.3% (**, p<0.01) for 1.2 mg/kg dosegroup.

Non-fasted blood glucose (mg/dL) in ob/ob mice administered vehiclecontrol or the fusion molecule at the indicated doses was assessed atvarious time points during the 22 day post-administration observationperiod. Referring to FIG. 2C, relative to vehicle control (PBS)-injectedob/ob mice (day 8=357.4 mg/dL), non-fasted blood glucose levels on day 8demonstrated a decrease of 3.9 mg/dl, which comprised a percent decreaseof 1.1% (ns) for 0.04 mg/kg dose group; a decrease of 62.7 mg/dL, whichcomprised a percent decrease of 17.5% (ns) for 0.12 mg/kg dose group; adecrease of 106.1 mg/dL, which comprised a percent decrease of 29.7% (*,p<0.05) for 0.40 mg/kg dose group; and a decrease of 191.1 mg/dL, whichcomprised a percent decrease of 53.5% (**, p<0.01) for 1.2 mg/kg dosegroup.

The data in FIG. 2 demonstrate that an HSA fusion with GDF15 is active,and that such fusion molecules represent a viable approach for enhancingcertain beneficial properties of GDF15 muteins. The data also indicatethat measurement of the indicated parameters may be useful as a platformfor high-throughput screening of muteins.

Example 2 Improvement of GDF15 Properties Via Reduction of SurfaceHydrophobicity

In an effort to identify means for improving the physical properties(e.g., solubility and stability) of mature human GDF15, a set of sixhydrophobic residues predicted to be surface-accessible were mutated toalanine as a means of increasing surface hydrophobicity.

Fusion molecules were generated wherein each of the six GDF15 muteinsequences was fused to HSA through the linker depicted in FIG. 1H (anon-cleavable 3×(4Gly-Ser) linker (SEQ ID NO:64); the sequences setforth in FIG. 3 neither depict the HSA component nor the linkercomponent of the fusion molecules.

The fusion molecules were then monitored for expression as secreteddisulfide-linked homodimers. FIG. 4 summarizes the data for each fusionmolecule. The first two columns identify the residue of the mature humanGDF15 that was mutated, the third column identifies those nativeresidues that were substituted by alanine, and the fourth columnindicates whether each resultant fusion molecule was expressed as asecreted dimer. Five of the six hydrophobicity-reduction muteinsexpressed and secreted as disulfide linked homodimers and were furtherevaluated for improvements in physical properties (w65 was not expressedas a homodimer and was not pursued further).

Example 3 Human GDF15 Muteins with Improved Solubility Characteristics

The data set forth in Example 2 were used to address solubilitylimitations associated with surface hydrophobicities inherent to maturehuman GDF15. In addition, the effect on solubility of introducingN-linked Glycosylation consensus site(s) along the sequence of maturehuman GDF15 was evaluated.

In order to facilitate assessment of solubility and determination of invivo efficacy, mature, recombinant human GDF15 and GDF15 muteins wereconstructed as N-terminal HSA fusion molecules containing a Factor Xaproteolytic-sensitive linker (a 2×(4Gly-Ser) Factor Xa cleavable linker(SEQ ID NO:56); as described in FIG. 1F) to allow for excision of theGDF15 or the GDF15 mutein from the HSA chaperone using Factor Xadigestion. Mature, recombinant human GDF15 and GDF15 muteins were alsoconstructed utilizing IgK as a signal peptide directly fused with themature 112 amino acid sequence of hGDF15 (FIG. 1B) or the muteinsequence of interest; or as bacterial refolds of muteins with anN-terminal methionine. Solubility assessments were performed in 1×PBS, astringent buffer for which improvements in the solubility of a muteincan be assessed relative to mature human GDF15 (which has a maximumobserved solubility of <0.2 mg/mL in 1×PBS).

Assessment of solubility was determined based on Absorbance at 280 nmusing Beer's law calculated using Extinction Coefficient (mature humanGDF15=14,400/monomer) and molecular weight (mature human GDF15=12,278Da/monomer). Muteins were categorized into one of five groups dependingon their level of solubility: 0.0-0.2 mg/mL=+; 0.2-0.5 mg/mL=++; 0.5-1.0mg/mL=+++; 1.0-5.0 mg/mL=++++; and >5.0 mg/mL=+++++.

Reduction of surface hydrophobicity of five of the GDF15 muteins (w29,w32, w52, w68 and w89) set forth in FIG. 5 was assessed via selectivemutagenesis of hydrophobic residues to alanine Comparison of therelative solubility of these five muteins to mature human GDF15indicated that w52 and w89 were the only muteins in this class thatexhibited improved solubility (++) relative to mature human GDF15 (+).The other three muteins exhibited solubility within the same range asmature human GDF15.

Reduction of the surface hydrophilicity of human GDF15 was assessed viaselective mutagenesis of acidic residues to alanine with the fivesequences denoted in FIG. 5 and summarized hereafter: w113, w114, w115,w116 and w117. Comparison of the relative solubility of these fivemuteins to mature human GDF15 indicated w116 was the only mutein in thisclass that exhibited improved solubility (++) relative to mature humanGDF15 (+). Of the other four muteins, w113 and w115 exhibited solubilitywithin the same range as mature human GDF15, while muteins w114 and w117were insoluble under the conditions employed.

Next, the mature human GDF15 sequence was assessed for its ability toaccommodate introduction of N-linked Glycosylation consensus site(s). Inthis context, a single amino acid substitution would impart the requiredconsensus site within the mature human GDF15 sequence, the consensussite for N-linked glycosylation being defined as “Asn-Xxx-Ser/Thr”,where “Xxx” cannot be a proline residue. Based on a scan of the maturehuman GDF15 sequence, 14 possible single-point muteins were identifiedthat would accommodate introduction of the N-Glycan consensus site. FIG.6 depicts the sequences of the 14 mono-glycosylation muteins, as well asadditional combinatorial di-Glycosylation muteins.

Before being assessed for solubility, each of the engineered N-Glycanmuteins set forth in FIG. 6 was evaluated both for secretion as a foldedGDF15 homodimer into mammalian tissue culture media and for N-glycansite occupancy. As set forth in FIG. 7, ten of the fourteenmono-glycosylated muteins were secreted as folded GDF15 homodimers,whereas muteins w123, w125, w127 and w129 did not result in dimerformation. The ten mono-glycosylation muteins that secreted ashomodimers were then assessed by LC/MS and SDS-PAGE gel shift todetermine occupancy of N-Glycan groups on the consensus site; two ofthese muteins (w121 and w124) exhibited low occupancy and theirsolubility was not subsequently evaluated.

Engineered N-Glycan GDF15 muteins which were both secreted as homodimersand possessed high glycan occupancy within the consensus site weremonitored for improvements to solubility relative to mature human GDF15.As denoted in FIG. 8A, each of the N-Glycan GDF15 muteins that wasassessed using the centrifugal assay format exhibited improvedsolubility compared to mature human GDF15: w118: +++; w120: ++++; w122:++; w126: ++++; w128: ++++; w130: ++++; w131: ++++; w132n: ++++; w133:++++; w134: ++++; w135: +++++; w136: ++++; w137: ++++; w138: +++++;w139: +++++; and w140: +++++. +++++.

As denoted FIG. 8B, each of the N-Glycan GDF15 muteins that was assessedusing the tubidity assay format exhibited improved solubility comparedto mature human GDF15: w120: ++++; w122: ++; w126: ++++; w128: ++++;w130: +++++; w131: ++++.

Acute in vivo efficacy was confirmed for those human mono-glycosylatedGDF15 muteins which displayed improved solubility compared to maturehuman GDF15.

Referring to FIG. 9A, following subcutaneous administration of a single0.3 mg/kg dose of mature human GDF15 or a N-glycan mutein to 7-15week-old male ob/ob mice (n=7), food intake (grams/animal) over a16-hour overnight period was monitored relative to a vehicle (PBS)control group. Three cohorts of mice were evaluated, and p-values weredetermined by an unpaired student T-test.

As shown in FIG. 9A, in the first cohort of mice, food intake decreasedan average of 0.82 g/animal (a percent decrease of 17.3% (***,p<0.001)), for the mature human GDF15 dose group; decreased 0.16g/animal (a percent decrease of 3.4% (ns)) for the w120 dose group;decreased 0.87 g/animal (a percent decrease of 18.3% (***, p<0.001)) forthe w128 dose group; decreased 0.77 g/animal (a percent decrease of16.2% (***, p<0.001)) for the w130 dose group; and decreased 0.45g/animal (a percent decrease of 9.4% (*, p<0.05)), for the w131 dosegroup. The average food intake for the vehicle control dose group was4.76 g/animal.

For the second cohort of mice shown in FIG. 9A, food intake decreased anaverage of 0.77 g/animal (a percent decrease of 17.1% (*, p<0.05)) forthe wild-type human GDF15 dose group; decreased 0.35 g/animal (a percentdecrease of 7.9% (ns)) for the w118 dose group; and decreased 0.59g/animal (a percent decrease of 13.0% (*, p<0.05)) for the w126 dosegroup. The average food intake for the vehicle control dose group was4.53 g/animal.

For the third cohort of mice shown in FIG. 9A, food intake decreased anaverage of 1.10 g/animal (a percent decrease of 23.4% (***, p<0.001))for the wild-type human GDF15 dose group; and decreased 1.29 g/animal (apercent decrease of 27.4% (***, p<0.001)) for the w122 dose group. Theaverage food intake for the vehicle control dose group was 4.70g/animal.

Referring to FIG. 9B, following an 8-hour fast, subcutaneousadministration of a single 1.0 mg/kg dose of PBS vehicle, mature humanGDF15 or a N-glycan mutein was given to 17 week-old male DIO mice (n=9).Food intake following refeeding (grams/animal) over a 16-hour overnightperiod was monitored relative to a vehicle (PBS) control group. P-valueswere determined by an unpaired student T-test. Referring to FIG. 9B,food intake decreases were monitored and are reported relative to PBSvehicle. For the mature human GDF15 dose group (***; p=0.0000003), w122(***; p=0.000006), w120 (***; p=0.00003), w118 (***; p=0.000001), w126(***; p=0.000008), w128 (ns; p>0.05), w130 (***; p=0.0000005) and w131(ns; p>0.05).

Example 4 Analytical Gel Filtration Analysis of Engineered N-GlycanGDF15 Muteins

Hydrodynamic radii of GDF15 N-Glycan muteins were assessed relative tomature human GDF15 utilizing analytical gel filtration chromatography(see FIG. 10). A TOSOH Biosciences TSKgelG3000SW_(XL) (7.8 mm ID×30 cm,5 μm) analytical sizing column pre-equilibrated in 1× phosphate bufferedsaline with flow rate of 1 mL/min was used in the evaluation. Two μg ofeach GDF15 mutein were injected in a 20 μL volume (0.1 mg/mL), andelution times were recorded at maximum absorbance during elution of thebell-shaped curve via measurement at 280 nm.

Analytical gel filtration chromatography of mature human GDF15 indicateda non-aggregated, disulfide-linked homodimer eluting at 10.837 minutes(FIG. 10). Each of the N-linked glycan muteins increased thehydrodynamic radii of the human GDF15 disulfide-linked dimer. Thus, eachmutein may potentially serve as a starting point for generatingmolecules having, for example, a favorable in vivo half-life.

Example 5 Expression of GDF15 Orthologs Utilizing HSA as Fusion Partner

The data set forth in Example 5 highlight the utility of HSA as a fusionpartner for expression and purification of GDF15 orthologs and other BMPfamily members.

FIG. 11A depicts the amino acid sequences of fusion molecules comprisingHSA having an IgK signal sequence (signal sequence underlined; SEQ IDNO:53) fused to the N-terminus of species orthologs of mature GDF15 Musmusculus (bold; SEQ ID NO:54) and Macaca mulatta (bold; SEQ ID NO:55)through a cleavable linker (bold and underlined; SEQ ID NO:56). FIG. 11Bdepicts the amino acid sequences of fusion molecules comprising HSAhaving an IgK signal sequence (signal sequence underlined; SEQ ID NO:53)fused to the N-terminus of mature human TGF-β1 (bold; SEQ ID NO:59) andmature human BMP2 (bold; SEQ ID NO:60) through a cleavable linker (boldand underlined; SEQ ID NO:61).

The profiles for mouse GDF15 (NP_035949.2) and Macaca GDF15 (EHH29815.1)mirrored that observed for human GDF15 in that they were expressed andpurified as folded homodimers in the context of a HSA fusion. Factor Xacleavage of the purified HSA fusion constructs and release/purificationof the GDF15 orthologs revealed homodimers with physical propertiesequivalent to that of the human sequence (data not shown). The homologyof the mature forms for each of mouse and Macaca GDF15 moleculesrelative to the human sequence is 67% and 95% respectively, indicatingthat the HSA fusion molecule template can accommodate sequence diversityin the GDF15 fusion partner.

The folding and secretion properties appeared to be altered for lowerhomology sequences relative to human GDF15 for family members such asthe BMP class and the TGF-β class of molecules. Indeed, when expressionwas attempted in the HSA template for secretion that was robust forhuman GDF15, mouse and Macaca GDF15; human BMP2 (27%% sequence identityrelative to human GDF15) and human TGF-β1 (22%% sequence identityrelative to human GDF15) displayed poor folding and secretion properties(FIG. 12).

The ability of human GDF15 to be expressed and secreted as a fullyfunctional and biologically active molecule can be accomplished using apolypeptide having 45% amino acid sequence identity to human GDF15(e.g., such as mouse and Macaca GDF15). The ability to express andpurify HAS fused to the N-Terminus of human GDF15 (or closely relatedmolecules) containing a linker of variable length and composition hasdirect implications for beneficial improvements to the pharmaceuticalproperties of GDF15 such as solubility, expression profile, formulationand stability.

Example 6 Identification of Residues Amenable to Mutagenesis withinHuman GDF15 to Allow for Engineering for Improved Physical Properties

A comprehensive Alanine scan was performed in which each amino acidwithin the mature sequence of human GDF15 was individually mutated toAlanine (with the exception of Cysteine residues so as to maintain thecysteine-knot structure/fold of GDF15). The sequences for each muteinare set forth in FIG. 13, and the expression results are detailed inFIG. 14. Each mutein described in FIG. 14 was purified and assessed forphysical properties, including homo-dimeric fold and aggregation state.

The results of the Alanine scan indicated that human GDF15 is amenableto mutagenesis at all residues with the exception of five muteins; w36,w46, w62, w65 and w83 (or additionally the introduction of a novelglycosylation consensus site(s) as defined in FIG. 7: w123, w125, w127or w129). All other muteins expressed, folded and secreted in a mannersimilar to that of wild-type human GDF15.

The identification of specific residues within GDF15 that canaccommodate mutagenesis as described herein aids in the design andengineering of a GDF15 molecule with improved physical properties. Theseproperties include, but are not limited to, correction of unwanted sitesof in vivo and/or in vitro proteolysis; and/or sites of chemicalheterogeneities such as deamidation, dehydration, succinimide formationand/or sites of oxidation; which may lead to drug inactivation and/ordecreased activity.

Experimentally defined chemical heterogeneities arising from acceleratedstability studies (data not shown) of mature sequence of human GDF15demonstrated evidence of deamidation events occurring at sites N3, Q40,Q51, N56, Q60, N84, Q90 and Q95; and oxidation events occurring at sitesW29, W32, M43, M57 and M86.

The results from the Alanine scan and identification of sites availablefor mutagenesis can be used to produce engineered constructs so as toimprove and correct the demonstrated chemical heterogeneities.

For example, the identification of sites amenable to mutageneis can beused to correct the deamidation event that occurs at N3 within themature sequence of GDF15. Having demonstrated that N3 is amenable tomutagenesis without detriment as illustrated by the N3A mutationdescribed above, it is reasonable to expect that other amino acids couldbe substituted at this site, such as N3Q, N3E, N3T or N3S. Amino acidssuitable for substitution can be identified by, for example, alignmentof human GDF15 with a non-human ortholog (e.g., Otolemur garnettii(XP_003796612.1; e.g., N3T) or Felis catus (XP_003982125.1; e.g., N3S).Finally, a deamidation event at N3 creates an unnatural Aspartateresidue at position 3, which could result in an iso-aspartateisomerization due to the presence of G4 directly C-terminal to thedeamidation event (i.e., Asp-Gly site). Deamidation at site N3 could beprevented by mutating the isomerization partner Gly to a Pro (G4P) todisrupt the Asp-Gly pairing. Additionally, deamidation can be reducedvia creation of an N-linked Glycosylation site at N3 via mutagenesis ofD5 to either D5T or D5S. Further, the deaminidation can be by truncationof the N-terminus of the mature GDF15 by removal of the first 3 residues(ΔA1-N3), 4 residues (ΔA1-G4), 5 residues (ΔA1-D5), 6 residues (ΔA1-H6)or more.

Based on the Alanine scanning mutagenesis results described above, andapplying similar analysis as applied to the site N3, correctivemutations to other observed sites of heterogeneity within the maturesequence of human GDF15 include, but are not limited to: N3A, N3Q, N3E,N3S, N3T, G4P, D5S, D5T, Q40A, Q40E, Q40D, Q40H, M43A, M43V, M43F, Q51A,Q51E, Q51L, Q51H, N56A, N56S, M57A, M571, M57T, Q60A, Q60L, N84A, N84E,N84Q, N84T, M86A, M86V, Q90A, Q90E, Q90E, Q90H, Q95A, Q95E, Q95D, Q95H,Q95T, Q95S.

The data set forth in Example 6 highlighted residues that are amenableto mutagenesis so as not to significantly impact folding or secretion ofGDF15 when expressed as a fusion protein comprising HSA fused to theN-terminus of mature GDF15 with a non-cleavable linker.

Example 7 Expression of Solubility Improved, Half-Life ExtendedMolecules Comprising N-Terminal Fusions of GDF15

The effect on solubility of introducing genetic fusions to theN-terminus of mature human GDF15 was evaluated. The data set forth inExample 7 were generated using several fusion constructs schematicallydepicted in FIG. 15A and having the amino acid sequences set forth inFIGS. 15B-15E.

FIG. 15B is a fusion molecule comprising a signal sequence(underlined)-Fc fused to the N-terminus of mature GDF15 (bold) through a3×(Glu-3Gly-Ser) linker (bold and underlined);

FIG. 15C is a fusion molecule comprising a signal sequence[underlined]-(Fc(+)GDF15/Fc(−) charged pair containingFc(+)-3×(Glu-3Gly-Ser)-GDF15 and Fc(−), wherein Fc(+)GDF15/Fc(−) chargedpairs were designed as a Fc(+)GDF15 fusion containing positive chargemutations in the Fc domain (D356K and D399K, underlined and bold in FIG.15C, which can be co-transfected with a Fc(−) domain containing negativecharge mutations in the Fc domain (K392D and K409D), underlined and boldin FIG. 15C.

FIG. 15D is a fusion molecule comprising an Albumin Binding Domain (ABD)fused to the N-terminus of GDF15 (bold) through a 5×Gly linker (bold andunderlined); the N-terminal methionine is underlined.

FIG. 15E is a fusion molecule comprising a signal sequence(underlined)-Maltose Binding Domain (MBD) fused to the N-terminus ofGDF15 (bold) through an enterokinase-cleavable 5×Gly linker (bold andunderlined).

Referring to the fusion constructs described above, those containing Fcand MBD were directly purified from transiently transfected 293 cellculture. ABD fusion was expressed and purified in a similar manner tothat of Mature GDF15 from bacterially expressed inclusion bodies.

In the case of the homo-dimeric Fc fusion construct (FcGDF15), theobserved secreted form was composed of high molecular weight oxidizedaggregates in the culture media. To circumvent this observedaggregation, a Fc(+)GDF15/Fc(−) charged pairs were designed as aFc(+)GDF15 fusion containing positive charge mutations in the Fc domain(D356K and D399K) that can be co-transfected with a Fc(−) domaincontaining negative charge mutations in the Fc domain (K392D and K409D).

Solubility assessments of ABD-GDF15, MBD-GDF15 and human GDF15 (control)were performed in 1×PBS, a stringent buffer for which improvements inthe solubility of a mutein can be assessed relative to mature humanGDF15 (which has a maximum solubility of <0.2 mg/mL). Assessment ofsolubility was determined based on Absorbance at 280 nm using Beer's lawcalculated using Extinction Coefficient and molecular weight for eachrespective fusion molecule. A280 Concentrations were confirmed withBradford protein quant at 595 nm relative to a BSA standard control.Fusion molecules were categorized into one of five groups depending ontheir level of solubility: 0.0-0.2 mg/mL=+; 0.2-0.5 mg/mL=++; 0.5-1.0mg/mL=+++; 1.0-5.0 mg/mL=++++; and >5.0 mg/mL=+++++. Each of theexpressed and purified fusion molecules that were assessed exhibitedimproved solubility compared to mature human GDF15: hGDF15: +;ABD-GDF15: +++++; MBD-GDF15: ++++(see FIG. 16 (Panel A)).

Reduction in body weight over 15 days was confirmed in vivo for the ABDfusion which displayed improved solubility compared to mature humanGDF15. Following subcutaneous administration of a single 3 mg/kg dose ofABD-GDF15 into 10 week-old male ob/ob mice (n=6), body weight(grams/animal) over a 15 day period was monitored relative to a vehiclecontrol group and p-values were determined by a two-way anova analysis(*=p<0.05; **=P<0.01; ***=p<0.001). In the case of ABD-GDF15, asignificant reduction in body weight was observed at day 15 indicating amolecule with extended efficacy. A follow-up PK profiling of theABD-GDF15 molecule in ob/ob mice at 0.3 mg/kg and 3 mg/kg dosesubcutaneously and monitored with time points taken over two weeks (datanot shown) demonstrated a T1/2 of 54.2 hrs (0.3 mg/kg) and 28.4 hrs (3mg/kg) (and see FIG. 16 (Panel B)).

Example 8 Expression of Platypus GDF15 Ortholog Recombinant Protein andEffects on Food Intake and Body Weight in DIO Mice

The data set forth in Example 8 exemplify the utility of constructionand expression of biologically active GDF15 orthologs using a signalpeptide and furin-cleavable pro-domain.

Mature Platypus GDF15 (Ornithorhynchus anatinus (Oa); AFV61279) wasrecombinantly produced as described herein using the construct depictedin FIG. 17A. Mature platypus GDF15 polypeptide exhibits ˜45% identity tomature human GDF15 polypeptide.

The effect on overnight food intake and body weight reduction wasdetermined in 17 week-old DIO mice (n=8) following a single,subcutaneous dose of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 and 1.0 mg/kg ofmature OaGDF15. Although mature platypus GDF15 polypeptide only exhibits45% sequence identity to mature human GDF15 polypeptide, a similar invivo effect was observed on food intake reduction (OaGDF15=0.03 mg/kg;hGDF15=0.04 mg/kg) and body weight reduction (OaGDF15=0.04 mg/kg;hGDF15=0.01 mg/kg) based on differences of pre-dose vs. 24 post-dose,respectively (see FIG. 17B (binding curves for human GDF15 are notshown).

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing, description, variations of the disclosedembodiments may become apparent to individuals working in the art, andit is expected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A dimer comprising two polypeptides, each of thetwo polypeptides comprising the amino acid sequence of SEQ ID NO:
 34. 2.The dimer of claim 1, wherein at least one of the two polypeptidescomprises an albumin fusion, wherein an albumin, an albumin variant, oran albumin fragment is conjugated to the at least one of the twopolypeptides.
 3. The dimer of claim 2, wherein the albumin, albuminvariant, or albumin fragment is human serum albumin, a human serumalbumin variant, or a human serum albumin fragment, respectively, orbovine serum albumin, a bovine serum albumin variant, or a bovine serumalbumin fragment, respectively, or cyno serum albumin, a cyno serumalbumin variant, or a cyno serum albumin fragment, respectively.
 4. Thedimer of claim 2, wherein the albumin, albumin variant, or albuminfragment is conjugated to at least one of the two polypeptides at thecarboxyl terminus or the amino terminus.
 5. The dimer of claim 4,wherein the albumin, albumin variant, or albumin fragment is conjugatedto at least one of the two polypeptides at the amino terminus.
 6. Thedimer of claim 4, wherein the albumin, albumin variant, or albuminfragment is conjugated to the at least one of the two polypeptides via alinker.
 7. The dimer of claim 6, wherein the linker is a cleavablelinker.
 8. The dimer of claim 7, wherein the cleavable linker can becleaved by a protease.
 9. The dimer of claim 7, wherein the linker is anon-cleavable linker.
 10. The dimer of claim 1, wherein the dimer isN-glycosylated.
 11. A transformed host cell that expresses the dimer ofclaim
 1. 12. A pharmaceutical composition, comprising the dimer of claim1, and a pharmaceutically acceptable diluent, carrier or excipient. 13.A pharmaceutical composition, comprising the dimer of claim 10, and apharmaceutically acceptable diluent, carrier or excipient.
 14. A sterilecontainer comprising the pharmaceutical composition of claim
 12. 15. Thesterile container of claim 14, wherein the sterile container is asyringe.
 16. A kit comprising the sterile container of claim
 14. 17. Amethod of treating obesity in a mammalian subject, the method comprisingadministering to the subject the dimer of claim 1, wherein the dimer isadministered in an amount effective in treating obesity in the subject.18. The method of claim 17, wherein the administering results inreduction in food intake by the subject.
 19. The method of claim 17,wherein the subject is a human and the administering results in areduction in body weight in the subject.
 20. The method of claim 17,wherein the subject is a human and the administering results in areduction in blood glucose in the subject.
 21. The method of claim 17,wherein the administering is by parenteral injection.
 22. The method ofclaim 21, wherein the parenteral injection is subcutaneous.
 23. A methodof treating hyperglycemia in a mammalian subject, the method comprisingadministering to the subject the dimer of claim 1, wherein the dimer isadministered in an amount effective in treating the hyperglycemia in thesubject.
 24. The method of claim 23, wherein the subject is human andthe administering results in reduction in blood glucose in the subject.25. The method of claim 23, wherein the administering results in areduction in body weight in the subject.
 26. The method of claim 23,wherein the administering results in a reduction in food intake by thesubject.
 27. The method of claim 23, wherein the subject has diabetesmellitus.
 28. The method of claim 23, wherein the subject is human. 29.The method of claim 23, wherein the subject is obese.
 30. The method ofclaim 23, wherein the administering is by parenteral injection.
 31. Themethod of claim 30, wherein the parenteral injection is subcutaneous.32. A method of treating obesity in a mammalian subject, the methodcomprising administering to the subject the dimer of claim 10, whereinthe dimer is administered in an amount effective in treating obesity inthe subject.
 33. The method of claim 32, wherein the administeringresults in reduction in food intake by the subject.
 34. The method ofclaim 32, wherein the subject is a human and the administering resultsin a reduction in body weight in the subject.
 35. The method of claim32, wherein the subject is a human and the administering results in areduction in blood glucose in the subject.
 36. The method of claim 32,wherein the administering is by parenteral injection.
 37. The method ofclaim 36, wherein the parenteral injection is subcutaneous.
 38. A methodof treating hyperglycemia in a mammalian subject, the method comprisingadministering to the subject the dimer of claim 10, wherein the dimer isadministered in an amount effective in treating the hyperglycemia in thesubject.
 39. The method of claim 38, wherein the subject is human andthe administering results in reduction in blood glucose in the subject.40. The method of claim 38, wherein the administering results in areduction in body weight in the subject.
 41. The method of claim 38,wherein the administering results in a reduction in food intake by thesubject.
 42. The method of claim 38, wherein the subject has diabetesmellitus.
 43. The method of claim 38, wherein the subject is human. 44.The method of claim 38, wherein the subject is obese.
 45. The method ofclaim 38, wherein the administering is by parenteral injection.
 46. Themethod of claim 45, wherein the parenteral injection is subcutaneous.